Mutant-enriched PCR Research Articles

Clinical implications of PNA‑sequencing as a complementary test for EGFR mutation analysis in human lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line regimen for the treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, false-negative results are occasionally observed, even with FDA-approved molecular tests. Such examples in have been reported in our pilot study showing a slightly upward-shifted amplification curve using commercial reverse transcription-quantitative (RT-q)PCR. Verification using peptide nucleic acid (PNA) clamping-sequencing, which has a sensitivity of ~0.1%, may allow better prediction of which patients will benefit from EGFR-TKI therapy. To confirm this hypothesis, samples were prospectively collected from 1,783 lung cancer cases diagnosed in National Cheng Kung University Hospital between 2012-2018. An independent lung cancer cohort of 1,944 cases was also recruited from other hospitals. The clinical significance of mutant-enriched PCR with PNA-sequencing was analyzed and patient outcomes were followed. A total of 17 of 34 cases (50%) were found to harbor EGFR mutations by PNA-sequencing. A total of 22 cases were discovered in the independent lung cancer cohort, and 14 of these (63.6%) cases had EGFR mutations. TKIs were administered to 14 of the 17 mutation-positive patients, and a partial response was observed in 4 cases and stable disease in 10 cases. Patients with EGFR mutations receiving a TKI regimen had a longer overall survival (OS) (median: 40.0 vs. 10.0 months) compared with those without treatment. The difference in OS was not significant. Based on the results of the present study, combining RT-qPCR with PNA-sequencing may be a practical supplementary technology in a clinical molecular laboratory for a subset of lung cancer patients in selection of EGFR TKI therapy.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Immunohistochemical Correlates of Epidermal Growth Factor Receptor Mutations in Lung Adenocarcinoma in Iraq Patient

We aimed to investigate the Detection of epidermal growth factor receptor (EGFR)mutations in lung adenocarcinoma by Immunohistochemical and correlation with Clinicopathological features. We used Fifty tissue blocks embedded in paraffin wax were obtained from 50 patient (16 female and 34males) suffering from lung adenocarcacinoma, while controls were 30 samples from healthy, Histopathological examination Study by (H and E stain)and detection of Mutation in EGFR(exon19) by RFLP-PCR, detection of protein expression EGFR by immunohistochemical method Our results have, confirmed the mean age of patients group was 51.84 +/-15.70 years and the median was 54, performed study the non correlation between stage of disease and age of patients. site of tumor was right sided of lung tumor, accounting for 58 % .Distribution of patients according to grade of tumor was, The results of this study showed non-significant correlation between age and grade, results of this study showed significant difference in EGFR Positive between patients and control healthy in Imunohistochemical, had found as not significant with age ,gender, sit of tumor, grade, stage, in Immunohistochemical method of EGFR Expression. while Gene EGFR deletion of exon 19were analyzed using mutant-enriched polymerase chain reaction (PCR) was not detected in all patients and control

Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.

Intratumor heterogeneity and tissue distribution of KRAS mutation in non-small cell lung cancer: implications for detection of mutated KRAS oncogene in exhaled breath condensate

PurposeMutated KRAS oncogene in exhaled breath condensate (EBC) can be a genetic marker of non-small cell lung cancer (NSCLC). However, a possibility of inhomogeneous distribution in cancer tissue and intratumor heterogeneity of KRAS mutation may decrease its significance. We investigated a status of KRAS point mutation and its sequence at codon 12 in 51 NSCLC patients after tumor resection. The comparison of KRAS mutation status between EBC–DNA and cancer tissue was performed in 19 cases.MethodsFive cancer tissue samples from disparate tumor regions and one from normal lung were harvested at surgery. EBC was collected for DNA analysis the previous day. KRAS point mutations at codon 12 were detected using mutant-enriched PCR technique and pyrosequenced.ResultsForty-six cancers revealed concordance of KRAS mutation status: 27 contained mutated KRAS and 19 had only wild KRAS. Five NSCLCs revealed inhomogeneous distribution of KRAS mutation. Two different mutations were found in 14 NSCLCs and the most frequent one was G12D and G12V (n = 8). No mutated KRAS was found in normal lung. The concordance ratios of KRAS sequence in codon 12 between EBC–DNA and cancer were 18/19 for NSCLC patients and 11/12 for KRAS mutation positive NSCLC.ConclusionsIntratumor heterogeneity and inhomogeneous distribution of KRAS point mutation in codon 12 in cancer tissue can occur in NSCLCs. There was a high accordance between KRAS mutation status in EBC–DNA and cancer tissue in NSCLC patients what suggests usefulness of monitoring KRAS mutation in EBC–DNA as a biomarker of NSCLC.

Evaluation of whole exome sequencing by targeted gene sequencing and Sanger sequencing

BackgroundTargeted gene sequencing (TGS) and whole exome sequencing (WES) are being used in clinical testing in laboratories. We compared the performances of TGS and WES using the same DNA samples. MethodsDNA was extracted from 10 endometrial tumor tissue specimens. Sequencing were performed with an Illumina HiSeq 2000. We randomly selected variants to confirm through Sanger sequencing or mutant-enriched PCR with Sanger sequencing. ResultsWe found that the variants identified in both TGS and WES were true positives (47/47), regardless of the sequencing depth. Most variants found in TGS only were true positives (34/40), and most of the variants found by WES only were false positives (8/18). From these results, we suggest that the sequencing depth may not play important role in the accuracy of NGS-based methods. After analysis, we found that WES had a sensitivity of 72.70%, specificity of 96.27%, precision of 99.44%, and accuracy of 75.03%. ConclusionsThe results of NGS-based methods must currently be validated, especially for important reported variants regardless of the methods used, and for the use of WES in cancers a higher false negative rate must be considered. More sensitive methods should be used to confirm the NGS results in uneven cancer tissues.

KRAS mutation testing on all non-malignant diagnosis of pancreatic endoscopic ultrasound-guided fine-needle aspiration biopsies improves diagnostic accuracy

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the procedure of choice for the cytologic diagnosis of pancreatic masses. The specificity of EUS-FNA approaches 100%, but the sensitivity is still low, and the high rate of indeterminate (atypical and suspicious) and false-negative results needs improvement. KRAS gene is frequently mutated in pancreatic ductal adenocarcinoma (PDAC) (up to 90%), and mutation analysis of KRAS has been proposed as diagnostic biomarker of PDAC. In most laboratories, KRAS mutation testing is performed by Sanger sequencing or real time-quantitative polymerase chain reaction (RT-qPCR), but these methods may give false-negative results in routine samples, mainly due to low cellularity. In order to increase the sensitivity of EUS-FNA, we propose a sequential approach for detecting KRAS mutations using mutant enriched-PCR (ME-PCR, sensitivity up to 0.1%) in cytologically indeterminate and negative samples tested wild-type by RT-qPCR. EUS-FNA specimens from 107 patients with pancreatic masses (51 males, 56 females, mean age 67 years) were cytologically examined. According to the Papanicolaou Society of Cytopathology guidelines, 50 cases (47%) were classified malignant, 15 (14%) suspicious, 13 (12%) atypical and 10 (9%) negative for malignancy; 18 cases (17%) were non-diagnostic. The overall specificity and sensitivity of cytological examination were 100% and 61%, respectively, when only negative and positive cases were considered; when atypical and suspicious were added to positive cases, the sensitivity increased to 95.1% and the specificity decreased to 85.7%. In all the cases, DNA was extracted from the cell-block and KRAS mutations were investigated by RT-qPCR, followed by ME-PCR in non-amplifiable and negative cases. The overall sensitivity and specificity of KRAS mutation testing alone were 79.3% and 100%; when KRAS mutation testing was performed in indeterminate and negative cytology, the sensitivity increased to 90% with specificity to 100%. Our data indicate that conventional cytology from EUS-FNA samples is highly specific for the diagnosis of pancreatic cancer. Indeterminate and negative cases need to be screened for KRAS mutations; this two-step approach may greatly improve the diagnostic accuracy of this method.

P3.02b-029 Primary Double EGFR Mutations T790M and Mutation in Exon 19 or 21 - Prevalence and Treatment Results in Slovakian NSCLC Patients: Topic: EGFR Biomarkers

Primary EGFR dual mutations comprising T790M and exon 19 or 21 mutation (SM, sensitizing mutations) are rare when common diagnostic methods are used. There are limited data about the treatment results with EGFR-TKIs in this setting. Purpose of this study was to find out the prevalence of primary dual EGFR mutations T790M and SM in the Caucasian population of NSCLC patients in Slovakia, and to evaluate treatment results with EGFR-TKIs in these patients. Retrospective multicenter study. The databases of the molecular/genetic diagnostic centers were searched for patients with dual EGFR mutations T790M and SM. Data regarding patients were obtained from the databases of participating institutions and patient files. Descriptive statistics was used for data analysis. Altogether 3883 patients were tested for EGFR mutation from 2010 through 2015. Allele specific PCR was used in majority, high resolution melting analysis, Sanger sequencing and mutant-enriched PCR were also used. Double mutations T790M and SM were found in only six cases, i.e. the observed period prevalence was 0.15%. Patients’ characteristics and the treatment results are in the Table. PS was improved after two months of treatment in patients with initial PS over 1, and remained unchanged in those with PS 1. There were no unexpected AEs. The prevalence of the dual EGFR mutations T790M and SM is very low in Caucasian population in Slovakia when common testing methods are used. Treatment results seen in this study suggest good effectiveness of the first or second generation EGFR-TKIs even in NSCLC with primary dual T790M and SM mutations. The quantitative analysis of these mutations using the blood sample is available at present. It might be useful in decision making about the use of the first – second or the third generation EGFR-TKI, based on the prevailing mutation.

The status of Her2 amplification and Kras mutations in mucinous ovarian carcinoma

Jayson GC et al. remarked in Lancet that nearly 100% of mucinous ovarian cancer cases have Kras mutation as well as a high frequency of Her2 amplification. Using the Abbott PathVysion Her2 DNA Probe Kit and Kras mutant-enriched PCR Kits (FemtoPath®), 21 samples of primary ovarian mucinous cystadenocarcinomas from Taiwanese patients were examined to determine the status of Her2 amplification and Kras mutations. Our results showed the Her2 amplification rates were 33.33%, while the Kras mutation rates were 61.90%. We present here our results in order to enlighten the readership that the ~100% Kras mutant frequency and the high Her2 amplification rate reported by Jayson et al. may be too exaggerated to be applicable into all populations. Additionally, we report another 2 novel Kras mutations (A11V, V14I).

Research Advancement on EGFR Mutation Detection of Cell-free DNA and Tumor Cell in Peripheral Blood of Patients with Non-small Cell Lung Cancer

非小细胞肺癌（non-small cell lung cancer, NSCLC）是肺癌中最常见的类型。表皮生长因子受体（epidermal growth factor receptor, EGFR）酪氨酸激酶受体抑制剂（tyrosine kinases inhibitors, TKIs）是目前最重要的靶向治疗药物，对于有EGFR敏感突变的NSCLC，采用EGFR-TKIs治疗能取得明显的临床疗效；T790M突变是最常见的EGFR-TKIs耐药机制，通过对外周血进行EGFR基因突变检测，可以筛选出EGFR-TKIs治疗有效或治疗过程中对其产生耐药的患者。其定量分析不仅在肿瘤的早期诊断中具有重要意义，而且是疗效评价及随访的重要生物学指标。目前，用于检测外周血EGFR突变的方法有很多，其中以在数字PCR基础上建立起来的ddPCR灵敏度最高，实现了对样本的高通量检测，定量程度较其他方法更为精确，在临床基因诊断中具有广阔的应用前景。

Abstract 2249: Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing

Abstract [Background] Mutational analysis is essential for recent personalized therapeutic approaches of non-small cell lung cancer (NSCLC) patients. Circulating cell-free DNA (cfDNA) from plasma is an emerging topic as a tool for liquid biopsy and possibly enable repetitive genetic diagnosis or monitoring of cancer status. However, detection of cfDNA from the circulation still remains challenging because of its highly fragmented and low concentration nature. In this study, we analyzed cfDNA samples by targeted deep sequencing at high coverage. [Materials and Methods] Twenty matched DNA samples were collected from metastatic NSCLC patients with EGFR mutation, which was detected by a clinical EGFR mutant enriched PCR assay using PNA-LNA PCR clamp method. These samples were extracted from tissue samples of fresh tumor tissue except one formalin fixed paraffin embedded (FFPE) sample (tDNA), and plasma samples (cfDNA). Mutation hotspots were enriched by multiplex PCR using the GeneRead DNAseq Targeted Panels V2 Human Tumor Actionable Mutations panel (QIAGEN, Hilden, Germany), followed by library construction, and sequenced on the MiSeq sequencer. Resulting data was analyzed using CLC Genomics Workbench and Genomics Server and mapped to the full human genome. [Results and discussion] The mean read depth in target region was 59,000 reads. Matched EGFR driver mutations between cfDNA and tDNA were detected with an overall concordance of 25% (5 of 20 samples). The concordance rate of exon21 L858R point mutation, exon19 deletions and exon20 T790M point mutation were 32.5% (3 of 8 samples), 16.7% (2 of 12 samples), and 66.7% (2 of 3 samples), respectively. The mutant allele frequencies in cfDNA ranged from 0.1% to 44.5% (median, 0.3%). While further improvement is mandatory, these results suggest that targeted deep sequencing of cfDNA at high coverage may have a potential utility as a non-invasive mutational analysis. In addition, there seemed to be a tendency of better detection rate for point mutations (45.5%) than deletions (16.7%), while the difference didn't reach statistical significance (p = 0.19), which were corresponding to previous reports. Citation Format: Ken Suzawa, Shuta Tomida, Takahiro Matsubara, Kadoaki Ohashi, Yuho Maki, Hiromasa Yamamoto, Mizuki Morita, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Katuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka. Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2249.

Circulating free DNA in a screening program for early colorectal cancer detection.

The quantification and molecular characterization of circulating free DNA (cfDNA) have attracted much interest as new and promising, noninvasive means of detecting and monitoring the presence of surgical resectable colorectal cancer (CRC). Instead, the role of cfDNA in the early detection of malignant and premalignant colorectal lesions is still unclear. The aim of this study was to evaluate the predictive power of the quantification and KRAS status of cfDNA in detecting early colorectal lesions in plasma from healthy high-risk subjects. The study population consisted of 170 consecutive healthy high-risk subjects aged >50 years who participated in the screening program promoted by the Local Health Service (ASL-Milano) for early CRC detection and who underwent endoscopic examination after being found positive at fecal occult blood test (FOBT). Thirty-four participants had malignant lesions consisting of 12 adenocarcinomas (at an early stage in half of the cases) and 22 instances of high-grade intraepithelial neoplasia (HGIN) in adenomas; 73 participants had premalignant lesions (adenomas and hyperplasia), and 63 participants had no lesions. Plasma cfDNA was quantified by quantitative real-time PCR and analyzed for KRAS mutations by a mutant-enriched PCR. KRAS status was assessed also in matched adenocarcinoma and HGIN tissues. The distribution of cfDNA concentrations among FOBT-positive subjects with diagnosed lesion (cases) was compared with that of FOBT-positive subjects without lesions (controls) and its predictive capability (AUC) was assessed. The predictive capability of cfDNA levels was satisfactory in predicting adenocarcinomas (AUC 0.709; 95% CI, 0.508-0.909) but not HGIN and premalignant lesions. The rate of KRAS mutations in plasma was low (5/170 = 3%) compared with the rate observed in the matched adenocarcinoma and HGIN tissues (45%). The use of cfDNA quantification to predict adenocarcinoma at an early stage in high-risk (aged >50 years and FOBT positive) subjects seems to be promising but needs more sensitive methods to improve cfDNA detection.

EGFR mutation status in plasma and tumor tissues in non-small cell lung cancer serves as a predictor of response to EGFR-TKI treatment

ABSTRACTObjective: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) can effectively control non-small cell lung cancer (NSCLC). Therefore, EGFR mutations should be detected before lung cancer patients undergo EGFR-TKI therapy. This study assessed the feasibility and predictive value of EGFR mutations in peripheral blood samples.Methods: EGFR mutations in exons 19 and 21 were analyzed in tumor tissue and plasma DNA samples from 121 NSCLC patients using amplification refractory mutation system (ARMS) and the integrated technique of mutant enriched PCR (me-PCR) and denaturing high performance liquid chromatography (DHPLC), respectively.Results: EGFR mutations were detected in 36.4% of tumor tissues and 34.7% of the plasma at a concordance rate of 85.1% (103/121). The sensitivity and specificity of plasma EGFR mutations were 77.3% and 89.6%, respectively. The gender and tumor histology of patients served as independent predictors of EGFR mutations in both tumor tissues and plasma, while CEA level was an independent predictor of EGFR mutations in the plasma. Furthermore, EGFR-TKI treatment showed a significantly higher objective response rate (ORR), median progression-free survival (mPFS), and overall survival (mOS) in patients harboring EGFR mutation than those that did not exhibit EGFR mutation (ORR: 69.4% versus 13.0% in tissues, P < 0.001; 64.5 % vs. 28.6% in the plasma, P = 0.006. mPFS: 10.4 months versus 4.1 months in tissues, P<0.001; 10.5 months vs. 5.2 months in the plasma, P=0.001. mOS: 25.7 months versus 8.3 months in tissues, P=0.005; 25.7 months vs. 13.5 months in the plasma, P=0.038).Conclusions: EGFR mutations can be detected in the plasma using the integrated technique of me-PCR and DHPLC, which enables us to predict patient response to EGFR-TKI therapy. High serum CEA levels served as an independent predictor for plasma EGFR mutations.

A New Fast and Highly Sensitive Assay for the Detection of Kras Mutations

ABSTRACT Aim: The demonstrated efficacy of anti-EGFR drugs (mAbs) in KRAS wild-type (wt) patients affected by colorectal cancer (CRC) resulted in increasing demand for mutation analyses. Mutation testing techniques have therefore become an urgent concern as a powerful mean to select wt patients. However, it is still unclear which is the most sensitive, specific and efficient protocol to detect KRAS mutations in clinical samples. The aim of this study was to compare the new Pentabase kit with direct sequencing (DS) (gold standard) and mutant-enriched PCR (ME-PCR) (highly sensitive method) with regard to sensitivity, specificity and efficiency of testing. Methods: Eight tumour cell lines, 7 specific for each KRAS mutation and 1 with the KRAS wt sequence, were used to perform a sensitivity assay. Seven different percentages of mutated DNA in fixed amounts of wt DNA were tested by all the methods (10%, 1%, 0,5%, 0,1%, 0,05%, 0,01% and 0%). KRAS mutations in exons 2 (codons 12 and 13) were evaluated by DS, ME-PCR and the Pentabase kit in a sensitivity assay and in 100 consecutive CRC patients. The Pentabase kit is based on real-time PCR using SuPrimers™ (DNA primers with increased specificity), BaseBlockers™ (oligos suppressing amplification of wt genes) and HydrolEasy probes (hydrolysis probe with increased signal-to-noise ratio and sensitivity). Results: In the cell line experiments, DS had a sensitivity of 10%, ME-PCR of up to 0,1% and the Pentabase kit of Conclusions: Overall, through the application of highly sensitive KRAS analysis methods, we detected additional KRAS alterations in up to 13/71 (18%) and 2/60 (3%) patients after DS and ME-PCR had shown them to be KRAS wt, respectively. The new kit allows the KRAS mutational typing in only 2 hours, compared to DS and ME-PCR, which require at least 2 days. Therefore the Pentabase kit represents a new, faster, more sensitive and more reliable method for the analysis of KRAS mutations in CRC and can be proposed for the identification of patients resistant to anti-EGFR mAbs. Disclosure: All authors have declared no conflicts of interest.

Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique

ObjectivesThe objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. Design and methodsWe developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. ResultsThe RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. ConclusionsCompared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.

Predictive factors associated with gefitinib response in patients with advanced non-small-cell lung cancer (NSCLC).

A number of different clinical characteristics have been reported to singly correlate with therapeutic activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). This study aimed to identify predictive factors associated with prognostic benefits of gefitinib. EGFR gene typing in 33 advanced NSCLC patients received gefitinib (250 mg/day) were analyzed with mutant-enriched PCR assay. Gefitinib response was evaluated with potential predictive factors retrospectively. The overall objective response rate (ORR) and median progression-free survival (PFS) in the 33 patients treated by gefitinib were 45.5% and 3.0 (2.0-4.0) months. The ORR and median PFS in EGFR gene mutation patients were significantly higher/longer than those in EGFR gene wild-type patients (P<0.01). Similarly, the ORR and median PFS in non-smoker patients were significantly higher/longer than those in smoker patients (P<0.05, P<0.01, respectively). However, no difference for ORR and median PFS occurred between male and female patients. Logistic multivariate analysis showed that only EGFR mutated gene was significantly associated with the ORR (P<0.01). Both EGFR mutated gene and non-smoker were the major factors that contributed to PFS (P<0.05). EGFR mutated gene and non-smoker status are potential predictors for gefitinib response in NSCLC patients.

Verification of Wild-Type EGFR Status in Non–Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases

EGFR genotyping is required for targeted therapy of lung adenocarcinoma. Because a false-negative result might prevent a patient from receiving appropriate targeted therapies, it is desirable to recheck equivocal results of EGFR genotyping. A cohort of 346 lung cancers was tested with a commercial kit for EGFR mutations; nine of the cases had upward real-time amplification curves at late cycles. They were also investigated using mutant-enriched PCR with peptide nucleic acid-locked nucleic acid (PNA-sequencing). Six of the nine equivocal cases harbored EGFR mutations. These cases likely had a small amount of mutant DNA near the detection limit of the commercial kit. Twenty nonequivocal, wild-type cases were reconfirmed using PNA-sequencing. We noticed a College of American Pathologists proficiency test material that showed a suspicious upward curve and eventually proved to have an H773_V774insPH in exon 20, for which a specific primer was not designed in the commercial kit. Further study using cloned DNA fragments showed that the upward curve most likely resulted from cross-reaction between similar, but nonidentical, sequences. It is desirable to keep the number of false-negative results as low as possible, but rechecking all wild-type cases is impractical. The late upward curves we observed helped identify suspicious cases for rechecking. A second method, such as PNA-sequencing, is recommended to verify wild-type cases.

580: Sensitive detection of NRAS mutations using mutant-enriched PCR and reverse-hybridization teststrips

580: Sensitive detection of NRAS mutations using mutant-enriched PCR and reverse-hybridization teststrips

FOLFOX-4 chemotherapy for patients with unresectable or relapsed peritoneal pseudomyxoma.

The standard treatment of peritoneal pseudomyxoma is based on cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC). The establishment of newer systemic treatments is an unmet clinical need for unresectable or relapsed peritoneal pseudomyxoma. The aim of our study was to assess the activity of chemotherapy with 5-fluorouracil and oxaliplatin (FOLFOX-4 regimen) in terms of response rate in this subset of patients. Patients were included in a single-center, observational study and treated with FOLFOX-4 administered every 2 weeks for up to 12 cycles or until progressive disease or unacceptable toxicity. Twenty consecutive patients were reviewed from July 2011 to September 2013. Only partial responses were observed, with an objective response rate of 20%. Median progression-free survival and overall survival were 8 months and 26 months, respectively. Two patients were able to undergo laparotomy with complete cytoreduction and HIPEC in one case. Safety data for FOLFOX-4 were consistent with the literature. By means of a mutant enriched polymerase chain reaction, KRAS mutation was found in 16 of 19 cases (84%), and MGMT promoter methylation was found in 8 (42%, all KRAS mutant). FOLFOX-4 chemotherapy is tolerable and active in patients with peritoneal pseudomyxoma when disease is deemed unresectable or relapsed after peritonectomy and HIPEC. The identification of predictive biomarkers, such as KRAS for resistance to anti-epidermal growth factor receptor monoclonal antibodies and MGMT for response to temozolomide, is a priority for the development of evidence-based treatment strategies for peritoneal pseudomyxoma.

Detection of KRAS codon 12 and 13 mutations by mutant-enriched PCR assay

BackgroundThe identification of KRAS mutations before the administration of anti-epidermal growth factor receptor (EGFR) therapy of metastatic colorectal cancer (mCRC) has become important. The aim of the present study was to develop a novel technology that can increase detection sensitivity for KRAS mutations. MethodsDNAs were extracted from colorectal cancer tissues and formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples. Mutant-enriched PCR assay utilizes the exceptionally thermostable endonucleases, PspGI for codon 12 and PhoI for codon 13, for specific amplifying KRAS mutations from mixed samples. The amplified PCR products were subjected to single-base primer extension or sequencing. Digital PCR was used to evaluate some of the results. ResultsWe compared the results with that from direct sequencing. In the FFPE samples, thirteen discordant samples were found. We showed that the mutant-enriched PCR assay can identify the codons 12 and 13 mutation in a mixed population of mutant and wild type DNA sequences at 1:1000 and 1:400, respectively. The sensitivity of this method is lower than the digital PCR. ConclusionsWe developed a rapid and highly sensitive method to detect codons 12 and 13 mutations of the KRAS gene. This method is a powerful tool for finding low-abundance variations in genomic DNA.