Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma

Purpose

Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported.

Methods

To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVision™ FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing.

Results

The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case.

Conclusion

IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas.     

高分辨率熔解曲线分析技术检测BRAF基因V600E突变方法的建立及初步临床应用

目的建立高分辨率熔解曲线分析技术(high resolution melting,HRM)检测BRAF基因V600E突变的方法,并探讨其在临床检测中的应用价值。方法用所建方法检测16例甲状腺乳头状癌患者超声引导下细针穿刺抽吸活检标本,并与测序法结果进行比较分析。结果所建HRM检测方法 Ct值与Tm的CV值均较小,重复性好。HRM法BRAF基因V600E突变检测标本的结果与测序法相比较,突变检出率分别为43.75%和40.00%,敏感性为100.00%,特异性为90.00%。结论成功建立的HRM法检测BRAF基因V600E突变敏感性高,特异性强,重复性好,操作简便,节约时间,成本低,适合细针穿刺抽吸活检标本检测BRAF基因V600E突变。     

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.     

Prevalence of BRAF V600E mutation in Chinese melanoma patients: large scale analysis of BRAF and NRAS mutations in a 432-case cohort

Background

Mutations of NRAS and BRAF have been described in Caucasian melanomas. However, the status and the clinical significance of BRAF and NRAS mutations in the Asian population have not been investigated on a large scale.

Methods

Melanoma samples (n = 432) were analysed for mutations in exons 11 and 15 of the BRAF gene, and exons 1 and 2 of the NRAS gene in genomic DNA by polymerase chain reaction (PCR) amplification and Sanger sequencing. Mutations of BRAF and NRAS genes were correlated to clinicopathologic features and prognosis of the patients.

Results

The incidence of somatic mutations within the BRAF and NRAS genes was 25.5% (110/432) and 7.2% (31/432), respectively. Among the 110 patients with BRAF mutations, 98 patients (89.1%) had V600E mutations. Melanomas without chronic sun-induced damage (Non-CSD) were more likely (P < 0.01) to show BRAF mutations while NRAS mutation frequency was unbiased between melanoma subtypes. Patients with genetic mutations in BRAF (P < 0.01) or NRAS (P = 0.04) gene are more likely to have ulceration as compared to patients without BRAF or NRAS mutations, respectively. Both BRAF (P = 0.003) and NRAS mutations (P = 0.031) are inversely correlated to overall survival.

Conclusions

BRAF mutation is frequent while mutations in NRAS gene are rare. The most prevalent BRAF mutation type is V600E. Patients with mutations in BRAF or NRAS gene are frequently present with ulceration, and mutation in BRAF or NRAS gene is indicator for poor prognosis. Our study may warrant a clinical trial of kinase inhibitors targeting BRAF V600E in Chinese and Asian melanoma patients.     

RUNX1 mutations in clonal myeloid disorders: from conventional cytogenetics to next generation sequencing, a story 40 years in the making

Translocations and mutations in the core binding factor genes, RUNX1 or CBFB, are found in acute myeloid and lymphocytic leukemia, therapy-related myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, and in familial platelet disorder with predisposition to acute myeloid leukemia. Here we review the biochemical and biological properties of the normal Runx1 protein, discuss the nature of RUNX1 mutations in myeloid leukemia, their prognostic significance, and the mutations that cooperate or co-exist with them in these various diseases.     

高分辨率熔解曲线分析技术检测EGFR基因突变方法的建立及其初步临床应用

目的建立高分辨率熔解曲线(high resolution melting,HRM)分析技术检测表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变的方法,并探讨其的临床应用价值。方法建立HRM技术检测EGFR基因突变方法,并用其检测200例非小细胞肺癌患者肿瘤石蜡包埋标本,并与测序法的结果进行比较。结果所建HRM检测方法 Tm与ct值的CV值均较小,重复性好。HRM法检测标本EGFR基因突变的结果与测序法相比,突变总检出率分别为40.0%和37.0%,敏感性为100%,特异性>95%。结论 HRM法检测EGFR基因突变,敏感性高,特异性强,重复性好,操作简便,节约时间,成本低,适用于临床。     

Comparison between p53 protein measurements using the luminometric immunoassay and immunohistochemistry with detection of p53 gene mutations using cDNA sequencing in human breast tumors

The p53mutational status of 226 representative primary breast cancer samples, derived from a population-based cohort, was analyzed using cDNA-based sequencing. The results were compared with those obtained with immunohistochemistry (IHC) on microwave-treated paraffin sections and the p53 specific luminometric immunoassay (LIA) on cytosols, all from the same individuals. Thirty-seven mutations were found using cDNA sequencing and were categorized into A) missense mutations in the evolutionarily conserved regions; B) missense mutations outside the evolutionarily regions; and C) deletions, insertions and nonsense mutations. Using optimal cut-off values, LIA detected 15 of 16 missense mutations in category A, in which IHC detected all 16. In category B, 10 of 13 and 7 of 13 mutations were detected, respectively. Some of the samples in category A had a very high p53 protein content when measured with the LIA, the reason for this being discussed. IHC detected 0 of 5 stop codon and 0 of 3 deletions/insertions mutations, while the LIA method detected 2 of 5 stop codon mutations and 1 of 3 deletion/insertion mutations. Compared with cDNA sequencing, protein analyses using optimal cut-off values resulted in an overall sensitivity and specificity of 64.9% and 89.9%, respectively, for the LIA method. Corresponding values were 72.2% and 92% for IHC. In addition, patients from whom p53 mutations could be detected by cDNA sequencing had a statistically significant (p = 0.0137) shorter survival, which was not readily apparent using the alternative LIA or IHC approaches at optimal cut-off values. Int. J. Cancer (Pred. Oncol.) 79:376–383, 1998. © 1998 Wiley-Liss, Inc.     

Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

Background  

Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.     

KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography

The Kirsten Rat Sarcoma (KRAS) oncogene has been introduced recently as a genetic biomarker for metastatic sporadic colorectal cancer prior to anti-EGFR treatment. Identifying patients with KRAS mutations that not respond to EGFR targeted therapies require sensitive, rapid and efficacious routine technique. We have attempted to evaluate the efficiency of three conventional methods: direct sequencing, HRM and DHPLC, to detect mutations in codon 12 and 13 of the KRAS exon2 gene. For this first Tunisian study on KRAS, we detected 45.83% of altered KRAS gene among 48 formalin-fixed paraffin-embedded sporadic colorectal adenocarcinoma patients. The use of HRM-sequencing allowed as enlarging the detected KRAS exon 2 mutations (22/48) in comparison with direct sequencing (17/48). DHPLC was used to confirm results when consensus was not observed between HRM and direct sequencing. This study brings an interesting data concerning an inter-method validation between sequencing and HRM in the investigation of sporadic colorectal cancer biomarker. It also shows that KRAS mutations occur at similar frequencies in Tunisian patients as in other populations; and suggests that the same genes are at play in sporadic CRC cancer, despite ethnic, geographical and environmental differences between countries.     

BRAF V600E mutation in metastatic colorectal cancer: Methods of detection and correlation with clinical and pathologic features

The screening for BRAF V600E mutation is employed in clinical practice for its prognostic and potentially predictive role in patients with metastatic colorectal carcinoma (mCRC). Little information is available on the sensitivity and specificity of the testing methods to detect this mutation in CRC. By using serial dilution of BRAF mutant DNA with wild type DNA, we found that the sensitivity of allelic discrimination-Real Time PCR was higher than PCR-Sequencing (10% vs 20%). In agreement, the Real Time PCR assay displayed increased analytical sensitivity in detecting the BRAF V600E mutation as compared with PCR-Sequencing in a cohort of 510 consecutive CRCs (21 vs 16 cases). Targeted resequencing demonstrated that all cases negative by PCR-Sequencing had an allelic frequency of the BRAF mutation <20%, thus suggesting tumor heterogeneity. The association of BRAF mutations with clinical and pathological features was assessed next in a cohort of 840 KRAS exon 2 wild type CRC patients screened with the Real Time PCR assay. The BRAF V600E mutation frequency in this cohort was 7.8% that increased to 33.4% in females over 70 y of age with right-sided tumor location. BRAF mutations were also detected in 4.4% of male patients with left-sided tumors and aged <70 y. Fourteen of 61 (22.9%) BRAF V600E mutation bearing patients exhibited microsatellite instability (MSI) as assessed by T17 mononucleotide sequence within intron 8 of HSP110. Our study indicates that Real Time PCR-based assays are more sensitive than PCR-Sequencing to detect the BRAF V600E mutation in CRC and that BRAF mutations screening should not be restricted to selected patients on the basis of the clinical-pathological characteristics.     

甲状腺癌超声表现与颈部淋巴结转移及BRAF V600E突变的关系研究

目的探讨甲状腺癌超声表现与颈部淋巴结转移以及BRAF V600E突变的关系。方法选取70例行甲状腺癌根治术及颈部淋巴结清扫的甲状腺癌患者,收集术前超声检查结果,术后行BRAF V600E基因突变检测。结果 70例患者中,颈部淋巴结转移率为30.00%,BRAF V600E突变率为71.43%;男性患者颈部淋巴结转移率明显高于女性患者,且超声表现为病灶多发、病灶直径﹥10 mm、边缘不规则、可接触被膜、血流丰富、钙化、病灶纵横比≥1患者的颈部淋巴结转移率均高于超声表现为病灶单发、病灶直径≤10 mm、边缘规则、不可接触被膜、血流不丰富、无钙化、病灶纵横比﹤1的患者,差异均有统计学意义(P﹤0.05);超声表现为病灶多发、边缘不规则、可接触被膜、血流丰富、钙化患者的BRAF V600E突变率均明显高于超声表现为病灶单发、边缘规则、不可接触被膜、血流不丰富、无钙化的患者,差异均有统计学意义(P﹤0.01)。Logistic回归分析结果显示,病灶多发、可接触被膜、血流丰富、钙化是发生颈部淋巴结转移的危险因素(OR=3.071、6.253、2.575、2.502,P﹤0.01);病灶多发是发生BRAF V600E突变的危险因素(OR=3.333,P﹤0.01)。结论甲状腺癌超声表现与颈部淋巴结转移、BRAF V600E突变有一定关系,值得进一步研究。     

Efficacy of first-line systemic treatment in correlation with BRAF V600E and different KRAS mutations in metastatic colorectal cancer - a single institution retrospective analysis

Background

KRAS mutation status in codons 12 and 13 is recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Despite having a wild type KRAS (wt-KRAS), not all patients with wt-KRAS respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway. Consequently, other molecular markers in colorectal cancer are needed to be evaluated to predict the response to therapy.

Patients and methods

In this retrospective study, objective responses (OR), time to progression (TTP), overall survival (OS) were analyzed in 176 metastatic colorectal cancer (mCRC) patients treated with first-line chemotherapy in combination with monoclonal antibodies in respect of KRAS status in codons 12 and 13 and BRAF mutational status.

Results

The KRAS mutations were found in 63 patients (35.8 %), the KRAS mutation in codon 12 in 53 patients (30.1%) and the KRAS mutation in codon 13 in 10 patients (5.7%). The BRAF V600E mutation was detected in 13 of 176 patients (7.4%). In the subgroup of mCRC patients having wt-KRAS and wild type BRAF (wt-BRAF), the objective response rates were higher (OR 54.0% ,CR 14.7%, PR 39.3%) than in the patients with wt-KRAS and mt-BRAF (OR 38.5%,CR 15.4%, PR 23.1%), the difference was not statistically significant (p= 0.378). Median OS in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 107.4 months and 45 months, respectively. The difference was statistically significant (p= 0.042). TTP in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 16 months and 12 months, respectively. The difference was not statistically significant (p= 0.558).

Conclusions

Patients with BRAF V600E mutation have statistically significantly worse prognosis than the patients with wt-BRAF and progress earlier during treatment. The definitive role of the BRAF V600E mutation as a prognostic and predictive factor for the response to anti-EGFR monoclonal antibodies needs to be analyzed in large prospective clinical studies.     

Association between BRAF V600E and NRAS Q61R mutations and clinicopathologic characteristics,risk factors and clinical outcome of primary invasive cutaneous melanoma

Purpose

Previous studies suggest that solar UV exposure in early life is predictive of cutaneous melanoma risk in adulthood, whereas the relation of BRAF mutation with sun exposure and disease prognosis has been less certain. We investigated the associations between BRAF^V600E and NRAS^Q61R mutations and known risk factors, clinicopathologic characteristics and clinical outcomes of melanoma in a case series of primary invasive cutaneous melanoma from the Nurses’ Health Study (NHS).

Methods

Somatic BRAF^V600E and NRAS^Q61R mutations of 127 primary invasive melanomas from the NHS cohort were determined by pyrosequencing using formalin-fixed, paraffin-embedded block tissues. Logistic regression analyses were performed to detect the associations of mutations with melanoma risk factors, and Kaplan–Meier method was used to examine associations between mutations and survival.

Results

The odds ratios for harboring BRAF^V600E mutations were 5.54 (95 % CI 1.19–25.8, p _trend = 0.02) for women residing in states with UV index ≥ 7 versus those residing in states with UV index ≤5 at 30 years of age. Patients with BRAF^V600E mutations tended to have shorter melanoma-specific survival when compared to patients with wild type at both loci (median survival time 110 vs. 159 months) (p = 0.03). No association was found between NRASQ61R mutation and melanoma risk factors or melanoma-specific survival.

Conclusions

BRAF^V600E mutations in primary cutaneous melanomas were associated with residence in locations with medium and high UV indices in mid-life. BRAF^V600E mutation may be associated with an unfavorable prognosis among melanoma patients.     

朗格汉斯细胞组织细胞增生症中 BRAF V600E 和 MAP2K1基因突变的分析及其临床意义

目的：探讨我国朗格汉斯细胞组织细胞增生症（Langerhans cell histiocytosis,LCH）中 BRAF V600E 和MAP2K1基因突变发生状况及其临床意义。方法：随机选取35例 LCH 组织标本,采用桑格测序法检测其中BRAF V600E 和 MAP2K1基因突变状况,免疫组化法检测 BRAF V600E 蛋白的表达。分析 BRAF V600E、MAP2K1基因突变与 LCH 临床基本资料（年龄、性别、单／多系统）的关系。结果：在35例 LCH 患者中,男女比例为1．7∶1,82．9％侵及骨组织,97．1％是单系统 LCH（single system LCH,SS －LCH）,2．9％是多系统 LCH （multi －system LCH,MS －LCH）。桑格测序法检测 BRAF V600E 基因突变率为17．1％,MAP2K1基因突变率为14．3％,MAP2K1与 BRAF V600E 基因突变有互异性;免疫组化法检测 BRAF V600E 阳性表达率为28．6％,涵盖了桑格测序法测得的突变病例。BRAF V600E 和 MAP2 K1基因突变更多出现在未成年组（35．7％和28．6％）,其中 BRAF V600E 突变在未成年人组与成人组间有显著性差异（P ＝0．028）;BRAF V600E 和MAP2K1基因突变对生存的影响无统计学差异（P ＞0．05）。结论：我国 LCH 患者大部分都是 SS －LCH,主要侵及的部位是骨组织,且预后良好,5年生存率为97．1％。桑格法所测的 BRAF V600E 和 MAP2K1基因突变率均低于西方报道,两者存在互异性,分别为17．1％和14．3％。所有 MAP2K1基因突变都是点突变,没有框内缺失突变,发现一个新的突变位点：c．112 G ＞A p．E38K;BRAF V600E 和 MAP2K1基因突变主要发生于未成年组中,提示各年龄层中 LCH 的发病机理可能不同,可能 RAS ／RAF ／MEK／ERK 通路在未成年人 LCH 中发挥更重要的作用;另外这两种突变对 LCH 的生存无影响。