KRAS Mutation Testing Research Articles

NRASQ61R Mutation-specific Immunohistochemistry is Highly Specific for Either NRASQ61R or KRASQ61R Mutation in Colorectal Carcinoma.

Anti-epidermal growth factor receptor-targeted therapy is only indicated in RAS wild-type colorectal carcinomas (CRCs). It is recommended that both NRAS and KRAS mutation testing to be performed before a CRC is considered RAS wild-type. Given that mutation-specific immunohistochemistry (IHC) has been shown to be sensitive and specific for the detection of NRAS mutations in melanoma, we assessed the specificity of NRAS mutation-specific IHC in CRC. IHC was performed on tissue microarrays containing 2823 consecutive CRC undergoing surgery with curative intent using a novel mutation-specific antibody to the protein produced by the NRAS mutation (clone SP174). Tissue microarrays were assessed by 2 observers and all IHC-positive or equivocal cases were repeated on whole sections to confirm the result. Positive cases then underwent molecular testing by matrix-assisted laser desorption/ionization-time of flight polymerase chain reaction. In total, 22 of 2823 (0.8%) CRCs demonstrated confirmed positive staining with complete interobserver concordance. RAS mutations were confirmed in all IHC-positive CRCs. In total, 11 cases harbored the NRASQ61R mutation. Surprisingly, 11 cases demonstrated the KRASQ61R mutation. We conclude that mutation-specific IHC with this currently available NRASQ61R antibody is highly specific for the presence of either NRASQ61R or KRASQ61R mutations in CRC. We caution that we did not assess the sensitivity of IHC and that this antibody does not detect other RAS mutations. Therefore, negative staining does not exclude a clinically significant RAS mutation. However, positive staining confirms the presence of an NRASQ61R or KRASQ61R mutation without the need for further molecular testing.

Abstract 4651: Multiple platform evaluation of KRAS codon 12/13 reference material

Abstract An assay’s ability to detect the presence of a KRAS driver mutation is critical when attempting to determine whether a tumor may respond to anti-EGFR therapy. Currently, there are two FDA approved companion diagnostics that can be used to assess whether there are mutations in KRAS codons 12 and 13, and both include their own internal controls. However, there is a need for external reference materials that allow laboratories to validate such assays - especially, at levels near their limits of detection (LODs). We describe the design of the Seraseq™ FFPE Tumor KRAS Reference Material. This reference material is composed of 7 different 5-micron FFPE curls that contain the 7 different KRAS mutations detected by the companion diagnostics: G12A, G12C, G12D, G12R, G12S, G12V and G13D. Each curl contains a mixture of KRAS wildtype GM24385 cells and a KRAS mutant cell line in sufficient quantity for most assays. KRAS mutant frequencies of about 5% were targeted and subsequently verified by digital PCR. A 5% mutant frequency was selected because it is near the LOD for the existing companion diagnostics, and is close to the minimum reported frequency of many next generation sequencing (NGS)-based assays that test for somatic variants. The reference material was evaluated using different methods, which include digital PCR, the Roche cobas® KRAS Mutation Test, the QIAGEN therascreen® KRAS RGQ PCR Kit and several NGS-based assays. Testing revealed general compatibility with these assays in that sufficient DNA could be extracted, and concordance of the results with the expected calls was high. For example, apparent DNA yields by Nanodrop with the cobas assay exceeded the required amount by more than 3-fold and all 7 curls led to a “mutation detected” result in duplicate testing. Interestingly, not all variants were detected with the therascreen assay, which may be due to some variants being present at slightly below the claimed LODs, however DNA yields were sufficient and the correct variants were identified. Finally, variant frequencies obtained by NGS were consistent with those observed by digital PCR. We conclude that the Seraseq FFPE Tumor KRAS Reference Material may be useful as part of KRAS codon 12/13 assay validation. Citation Format: Russell Garlick, Yves Konigshofer, Farol L. Tomson, Dale Yuzuki, Bharathi Anekella. Multiple platform evaluation of KRAS codon 12/13 reference material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4651. doi:10.1158/1538-7445.AM2017-4651

Abstract 5688: Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients

Abstract Background: Mutations in the KRAS proto-oncogene, GTPase (KRAS) are driver alterations in several tumors, such as non-small-cell lung (NSCLC) and colorectal cancer (CRC). However, their role as a prognostic marker in liquid biopsies is unclear. We studied the status of KRAS mutations in the peripheral blood of advanced NSCLC and colorectal cancer patients visited in our hospital and evaluated its potential value as a monitoring marker in the clinical practice. Methods: We developed a sensitive TaqMan assay, in the presence of a PNA clamp, for the determination of KRAS mutations (codons 12, 13 and 61) in circulating-free DNA (cfDNA). The assay detected 2.5 pg mutated DNA/µL and a ratio of KRAS mutated versus wild type allele of 1:20000 and was validated in 80 cancer patients previously genotyped in tumor tissue showing a clinical sensitivity of 72.5% and specificity of 100%. cfDNA was isolated from serum and plasma specimens, using an automatic extractor and mutational analyses were performed in quadruplicates samples. Serum and plasma samples were drawn at diagnosis, during follow-up and progression. Results: 239 NSCLC and 11 colorectal cancer patients were screened for KRAS mutations in cfDNA at presentation in the period of 2013 to 2016. In addition, 160 cfDNA samples collected during disease monitoring of 61 patients were analyzed. Paired biopsies were available from 110/250 p at diagnosis (60 positive for KRAS in FFPE and 50 WT). The KRAS mutation was detected in the cfDNA of 76.6% (46/60) p positive in tumor tissue vs. only in 1/50 WT patients where a G12C was detected in cfDNA in a consistent manner, probably associated with a heterogeneity of the tumor. In patients with no biopsy available, we detected KRAS mutations in cfDNA in 14.3% (20/140) cases at diagnosis. The most prevalent mutations observed in cfDNA were G12C (44.7 %), G12V (22.4%) and G12D (10.4%) for NSCLC patients and G13D (5.9%) for colorectal patients. The Q61H mutation was detected in the cfDNA of one NSCLC and one CRC p. Of the 67 patient’s positive in cfDNA at presentation, KRAS mutations were detected in serum and plasma in 38 patients, only in plasma in 24 and only in serum in 5. Regarding the 61 patients with serial blood samples, we observed a correlation between the KRAS mutation load in cfDNA and the course of the disease, with a decrease in patients responding to chemotherapy and an increase during disease progression. Conclusions: Our results demonstrate the feasibility of KRAS mutation analysis in the cfDNA of advanced NSCLC and colorectal patients. Analysis of KRAS mutations in the blood of mutated patients can have a value in patients with no biopsy available and to monitor the course of the disease. Citation Format: Mónica Garzón Ibañez, Clara Mayo de las casas, Nùria Jordana Ariza, Ariadna Balada Bel, Beatriz Garcia, Sergio Villatoro, Jordi Bertrán-Alamillo, Alejandro Martinez-Bueno, Santiago Viteri Ramirez, Ana Pérez Rosado, Daniela Morales Espinosa, Maria José Catalán, Niki Karachaliou, Miguel Ángel Molina-Vila, Rafael Rosell. Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5688. doi:10.1158/1538-7445.AM2017-5688

Abstract 1761: A rapid and accurate nucleic acid amplification and detection method for KRAS mutation testing in colorectal cancer specimens

Abstract Colorectal cancer (CRC) is one of the most common type of cancers both men and women in worldwide. Fortunately, overall death rates of CRC have been decreasing for the last two decades due to the improvement of screening test assays that detect early-stage cancer and pre-cancerous polyps. Nevertheless, the most common treatment for CRC is surgery, because it may completely eliminate the cancer region. In case of the cancer with systemic metastasis, chemotherapy is required before or after surgery for primary or metastatic lesions. Among the regimens for the chemotherapy, both monoclonal antibodies (Cetuximab and Pantitumumab) against the epidermal growth factor receptor have been shown to improve survival for only patients with lack of RAS mutations. Thus, the KRAS gene mutations (codons 12 and 13) in CRC patients have been extensively studied as a strong negative predictive biomarker to indicate whether a CRC patient responds to the treatment. Therefore, testing the KRAS mutational status of tumor samples is becoming an essential tool for managing patients with CRCs. Although a myriad of nucleic acid testing methods have been developed to analyze the mutation status in the key regions of the KRAS gene of CRC, several obstacles still remain related to low sensitivity, time consuming, and required large instruments including thermal cyclers. Here, we present a novel nucleic acid amplification and detection method for KRAS mutations (G12D and G13D) testing that enable rapid and accurate detection. This method is based on combination of isothermal DNA amplification method and bio-photonic silicon sensor that can be detected the mutations in a label-free and real-time manner. The proposed method can detect the mutant cell present at 1% in a mixture of wild type cells, while both PCR and sequencing can detect the mutations in a sample containing approximately 30% of mutant cells. We used 60 tissue samples from CRC patients (22 samples with G12D mutations, 23 samples with G13D mutation, and 15 samples with no mutation) to compare the clinical utility of three methods including PCR, Sanger sequencing and the proposed method. The proposed method with both G12D and G13D showed a value of 100% and 100% for sensitivity and specificity, respectively. One the other hand, the sensitivity and specificity of PCR (90.5% and 100%) and sequencing (95% and 100%) were lower than that of the proposed method. Therefore, the proposed method was found to be a rapid (< 30 min), highly sensitive and specific method for KRAS mutation testing. We believe that this rapid and accurate method will enable proper treatment for CRC patients. Note: This abstract was not presented at the meeting. Citation Format: Yong Shin, Choong Eun Jin, Seung-Seop Yeom, Seok-Byung Lim. A rapid and accurate nucleic acid amplification and detection method for KRAS mutation testing in colorectal cancer specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1761. doi:10.1158/1538-7445.AM2017-1761

Abstract 748: A rapid and accurate nucleic acid amplification and detection method for KRAS mutation testing in colorectal cancer specimens

Abstract Colorectal cancer (CRC) is one of the most common type of cancers both men and women in worldwide. Fortunately, overall death rates of CRC have been decreasing for the last two decades due to the improvement of screening test assays that detect early-stage cancer and pre-cancerous polyps. Nevertheless, the most common treatment for CRC is surgery, because it may completely eliminate the cancer region. In case of the cancer with systemic metastasis, chemotherapy is required before or after surgery for primary or metastatic lesions. Among the regimens for the chemotherapy, both monoclonal antibodies (Cetuximab and Pantitumumab) against the epidermal growth factor receptor have been shown to improve survival for only patients with lack of RAS mutations. Thus, the KRAS gene mutations (codons 12 and 13) in CRC patients have been extensively studied as a strong negative predictive biomarker to indicate whether a CRC patient responds to the treatment. Therefore, testing the KRAS mutational status of tumor samples is becoming an essential tool for managing patients with CRCs. Although a myriad of nucleic acid testing methods have been developed to analyze the mutation status in the key regions of the KRAS gene of CRC, several obstacles still remain related to low sensitivity, time consuming, and required large instruments including thermal cyclers. Here, we present a novel nucleic acid amplification and detection method for KRAS mutations (G12D and G13D) testing that enable rapid and accurate detection. This method is based on combination of isothermal DNA amplification method and bio-photonic silicon sensor that can be detected the mutations in a label-free and real-time manner. The proposed method can detect the mutant cell present at 1% in a mixture of wild type cells, while both PCR and sequencing can detect the mutations in a sample containing approximately 30% of mutant cells. We used 60 tissue samples from CRC patients (22 samples with G12D mutations, 23 samples with G13D mutation, and 15 samples with no mutation) to compare the clinical utility of three methods including PCR, Sanger sequencing and the proposed method. The proposed method with both G12D and G13D showed a value of 100% and 100% for sensitivity and specificity, respectively. One the other hand, the sensitivity and specificity of PCR (90.5% and 100%) and sequencing (95% and 100%) were lower than that of the proposed method. Therefore, the proposed method was found to be a rapid (< 30 min), highly sensitive and specific method for KRAS mutation testing. We believe that this rapid and accurate method will enable proper treatment for CRC patients. Note: This abstract was not presented at the meeting. Citation Format: Choong Eun Jin, Seung-Seop Yeom, Yong Shin, Seok-Byung Lim. A rapid and accurate nucleic acid amplification and detection method for KRAS mutation testing in colorectal cancer specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 748. doi:10.1158/1538-7445.AM2017-748

Genomic profiling of colorectal cancers and the future of personalized treatment

New technologies have enabled faster, cheaper and more accurate genomic and other types of profiling. Therefore, treatment has become more customized according to molecular subtype. Here, we summarize the current status of genomic profiling for colorectal cancer (CRC) and discuss future directions. Recently, the CRC Subtyping Consortium classified CRC into four subtypes: CMS1, microsatellite instability immune (14%); CMS2, canonical (37%); CMS3, metabolic (13%); and CMS4, mesenchymal (23%). Testing for KRAS, NRAS and BRAF mutations, and microsatellite instability status in CRC has proven essential for treatment decisions. Tumor heterogeneity and the evolution of drug-resistant subclones after therapy should be further assessed and pursued. Patient-derived xenografts and liquid biopsies might facilitate the development of optimum and accurate personalized therapy regimens.

SensiScreen®KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations.

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase’s proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25–1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods.

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.

Clinically Viable Gene Expression Assays with Potential for Predicting Benefit from MEK Inhibitors.

Purpose: To develop a clinically viable gene expression assay to measure RAS/RAF/MEK/ERK (RAS-ERK) pathway output suitable for hypothesis testing in non-small cell lung cancer (NSCLC) clinical studies.Experimental Design: A published MEK functional activation signature (MEK signature) that measures RAS-ERK functional output was optimized for NSCLC in silico NanoString assays were developed for the NSCLC optimized MEK signature and the 147-gene RAS signature. First, platform transfer from Affymetrix to NanoString, and signature modulation following treatment with KRAS siRNA and MEK inhibitor, were investigated in cell lines. Second, the association of the signatures with KRAS mutation status, dynamic range, technical reproducibility, and spatial and temporal variation was investigated in NSCLC formalin-fixed paraffin-embedded tissue (FFPET) samples.Results: We observed a strong cross-platform correlation and modulation of signatures in vitro Technical and biological replicates showed consistent signature scores that were robust to variation in input total RNA; conservation of scores between primary and metastatic tumor was statistically significant. There were statistically significant associations between high MEK (P = 0.028) and RAS (P = 0.003) signature scores and KRAS mutation in 50 NSCLC samples. The signatures identify overlapping but distinct candidate patient populations from each other and from KRAS mutation testing.Conclusions: We developed a technically and biologically robust NanoString gene expression assay of MEK pathway output, compatible with the quantities of FFPET routinely available. The gene signatures identified a different patient population for MEK inhibitor treatment compared with KRAS mutation testing. The predictive power of the MEK signature should be studied further in clinical trials. Clin Cancer Res; 23(6); 1471-80. ©2016 AACRSee related commentary by Xue and Lito, p. 1365.

Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples

BackgroundTesting for KRAS mutations in metastatic colorectal cancer (mCRC) on formalin-fixed, paraffin embedded (FFPE) tumor tissue has become standard of care. Different molecular methods exist to determine hotspot KRAS mutations in exon 2, 3 and 4, but testing is often limited by the sensitivity and the speed of analysis. The aim of this retrospective study was to establish the clinical performance of the Idylla™ KRAS Mutation Test on FFPE tumor samples of patients with mCRC.MethodsKRAS mutation analysis was performed using the therascreen KRAS on the RotorGene Q platform (CE-IVD; Qiagen) and results were subsequently compared to the Idylla™ KRAS Mutation Test. Discordant result testing was performed with massive parallel sequencing or alternative routine approaches.ResultsData from 182 samples were used to show that the overall agreement between the two methods for mutation characterization was 96.7% [95%CI: 93.0%-98.5%]. Six out of 182 samples (3.3%) showed true discordant results.ConclusionThe Idylla™ KRAS Mutation Test allows for a fast and reliable analysis of FFPE samples with a turnaround-time of two hours without the need of molecular infrastructure or expertise in order to guide the personalized treatment of colorectal cancer patients.

P2.03b-031 Impact of PD-L1 Status on Clinical Response in SELECT-1: Selumetinib + Docetaxel in KRASm Advanced NSCLC: Topic: Biomarkers

Anti-PD-1/PD-L1 immunotherapy has delivered clinical benefit for patients with NSCLC, and PD-L1 has emerged as a predictive biomarker. In the Phase III SELECT-1 trial (NCT01933932), selumetinib (AZD6244, ARRY-142886), an oral, potent and selective, allosteric MEK1/2 inhibitor with a short half-life, plus second-line docetaxel did not provide clinical benefit for patients with KRAS-mutant (KRASm) NSCLC compared with placebo plus docetaxel (PBO+DOC). Although no incremental benefit was observed, it is important to evaluate biomarkers, such as PD-L1, to understand more about the biology of patients with KRASm NSCLC. In total, 510 patients with a prospectively, centrally confirmed KRAS mutation (cobas® KRAS Mutation Test, Roche Molecular Systems) were randomized 1:1 to selumetinib 75 mg BID, plus docetaxel 75 mg/m2 q21d (SEL+DOC), or PBO+DOC. Evaluations included progression-free survival (PFS) by investigator assessment (RECIST 1.1; primary endpoint), and overall survival (OS). Association of tumor PD-L1 status with clinical responses was assessed as an exploratory objective. PD-L1 status was centrally determined using the PD-L1 IHC 28-8 pharmDx test (Dako) for all patients with sufficient tumor sample. Samples with a pre-specified cut-off of ≥5% tumor cell staining were considered PD-L1 positive. SEL+DOC did not improve PFS or OS compared with PBO+DOC. PD-L1 status was determined for 385 (75%) patients: 224 (58%) samples were PD-L1 <5%, and 161 (42%) samples were PD-L1 ≥5%; the remaining 125 patients had unknown PD-L1 status due to insufficient tumor sample. Subgroups were balanced across treatments. PD-L1 subgroup analysis of PFS and OS is presented below.Tabled 1SubgroupEvents (%) in SEL+DOC groupEvents (%) in PBO+DOC groupHR (95% CI)PFSPD-L1 <5%94/112 (84%)101/112 (90%)0.89 (0.67, 1.18)PD-L1 ≥5%65/79 (82%)71/82 (87%)0.70 (0.50, 0.99)PD-L1 unknown59/63 (94%)57/62 (92%)1.24 (0.86, 1.79)OSPD-L1 <5%73/112 (65%)74/112 (66%)0.94 (0.68, 1.30)PD-L1 ≥5%55/79 (70%)58/82 (71%)0.89 (0.61, 1.28)PD-L1 unknown48/63 (76%)38/62 (61%)1.57 (1.02, 2.41) Open table in a new tab Prevalence of PD-L1 positive status in this KRASm cohort was similar to that reported for a pan-NSCLC cohort (Borghaei, NEJM 2015). No significant PFS or OS differences were observed between treatments in either PD-L1 positive or negative tumors. Additional biomarker analyses are planned for different KRAS codon mutations, and LKB1 and TP53 status.

P2.03b-053 Role of KRAS Mutation Status in NSCLC Patients Treated on SWOG S0819, a Phase III Trial of Chemotherapy with or without Cetuximab: Topic: Biomarkers

The S0819 phase III study of chemotherapy and bevacizumab (by patient/physician choice) with or without cetuximab in NSCLC showed no benefit from the addition of cetuximab, either overall or within the EGFR FISH-positive subset. Secondary analysis suggested an overall survival benefit in EGFR FISH-positive squamous cell carcinoma (SCC) (Herbst WCLC 2015, Hirsch ASCO 2016). In colorectal cancer (CRC), benefit from EGFR monoclonal antibodies such as cetuximab is limited to patients with RAS wild type (WT) tumors; however, in NSCLC, previous studies have not been sufficiently powered to make this determination. We prospectively incorporated KRAS mutation testing in S0819 to determine whether it predicts cetuximab efficacy. Since KRAS mutations are rare in SCC, we focused this analysis on nonSCC. KRAS mutation status was determined using the Therascreen KRAS test (Qiagen), conducted in a CLIA-certified diagnostic laboratory at the UC Davis Comprehensive Cancer Center. This test is FDA-approved for KRAS diagnostics in metastatic CRC, and identifies 6 mutations at codon 12 (G12A,D,R,C,S,V) plus G13D. KRAS mutation status was available for 448 nonSCC patients, and mutations were identified in 150 cases (33%). Amino acid substitutions matched the expected distribution for a NSCLC population, with 52% harboring G12C and 17% with G12V. No significant differences were observed between KRAS-mut and WT populations for PFS (HR=1.15 (0.94-1.42); p=0.18) or OS HR=1.10 (0.89-1.37); p=0.39). Furthermore, no differences in outcomes between arms were observed based on KRAS mutation status (Table). The KRAS WT, EGFR FISH+ molecular subset (hypothetically the most likely subgroup to benefit from cetuximab) showed no statistical differences in outcomes between arms. Determination of KRAS mutation status did not identify a subgroup of nonSCC patients with differential outcome from addition of cetuximab to front-line chemotherapy. In contrast to CRC, cetuximab does not appear to confer benefit to patients with KRAS-WT nonSCC NSCLC.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay.

BackgroundMutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment.MethodsWe compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing.ResultsKRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%.ConclusionsThe cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Endoscopic ultrasound-guided fine-needle aspiration plus KRAS and GNAS mutation in malignant intraductal papillary mucinous neoplasm of the pancreas.

Background: KRAS and GNAS mutations are common in intraductal papillary mucinous neoplasia of the pancreas (IPMN). The aims of this study were to assess the role of pre-therapeutic cytopathology combined with KRAS and GNAS mutation assays within cystic fluid sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to predict malignancy of IPMN. Patients and methods: We prospectively included 37 IPMN patients with clinical and/or imaging predictors of malignancy (men: 24; mean age: 69.5 years). Cytopathology (performed on cystic fluid and/or IPMN nodules), KRAS (Exon 2, codon 12) and GNAS (Exon 8, codon 201) mutations assays (using TaqMan® allelic discrimination) were performed on EUS-FNA material. The final diagnosis was obtained from IPMN resections (n = 18); surgical biopsies, EUS-FNA analyses, and follow-up (n = 19): 10 and 27 IPMN were benign and malignant, respectively. Results: Sensitivity, specificity, positive and negative predictive values, and accuracy of cytopathology alone to diagnose IPMN malignancy were 55 %, 100 %, 100 %, 45 %, and 66 %, respectively. When KRAS-mutation analysis was combined with cytopathology these values were 92 %, 50 %, 83 %, 71 %, and 81 %, respectively. GNAS assays did not improve the performances of cytopathology alone or those of cytopathology plus a KRAS assay. Conclusions: In patients with a likelihood of malignant IPMN at pre-therapeutic investigation, testing for KRAS mutations in cystic fluid sampling by EUS-FNA improved the results of cytopathology for the diagnosis of malignancy whereas GNAS mutation assay did not.

Erratum to: PNA clamping-assisted fluorescence melting curve analysis for detecting EGFR and KRAS mutations in the circulating tumor DNA of patients with advanced non-small cell lung cancer.

Circulating cell-free DNA (cfDNA) is emerging as a surrogate sample type for mutation analyses. To improve the clinical utility of cfDNA, we developed a sensitive peptide nucleic acid (PNA)-based method for analyzing EGFR and KRAS mutations in the plasma cfDNA of patients with advanced non-small cell lung cancer (NSCLC). Baseline tissue and plasma samples were collected from treatment-naive advanced NSCLC patients participated in a randomized phase II study, which was registered with ClinicalTrials.gov at Feb. 2009 (NCT01003964). EGFR and KRAS mutations in the plasma cfDNA were analyzed retrospectively using a PNA clamping-assisted fluorescence melting curve analysis. The results were compared with those obtained from tissue analysis performed using the direct sequencing. Exploratory analyses were performed to determine survival predicted by the plasma and tissue mutation status. Mutation analyses in matched tissue and plasma samples were available for 194 patients for EGFR and 135 patients for KRAS. The mutation concordance rates were 82.0 % (95 % confidence interval [CI], 76.5–87.4) for EGFR and 85.9 % (95 % CI, 80.1–91.8) for KRAS. The plasma EGFR mutation test sensitivity and specificity were 66.7 % (95 % CI, 60.0–73.3) and 87.4 % (95 % CI, 82.7–92.1), respectively, and the plasma KRAS mutation test sensitivity and specificity were 50.0 % (95 % CI, 41.6–58.4) and 89.4 % (95 % CI, 84.2–94.6), respectively. The predictive value of the plasma EGFR and KRAS mutation status with respect to survival was comparable with that of the tissue mutation status. These data suggest that plasma EGFR and KRAS mutations can be analyzed using PNA-based real-time PCR methods and used as an alternative to tumor genotyping for NSCLC patients when tumor tissue is not available.

94P - An extended KRAS mutation test for the detection of 28 common mutations in FFPET and plasma specimens

94P - An extended KRAS mutation test for the detection of 28 common mutations in FFPET and plasma specimens

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer.

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients.

Detection of KRAS mutations of colorectal cancer with peptide-nucleic-acid-mediated real-time PCR clamping

ABSTRACTColorectal cancer (CRC) is the third most common cancer in the world and its disease-specific mortality is estimated to be approximately 33% in the developed world. KRAS mutations have been shown to predict response to anti-EGFR (epidermal growth factor receptor) targeted monoclonal antibody therapy. Therefore, KRAS mutation testing of metastatic CRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Currently, the most common strategy for KRAS mutation detection consists of conventional polymerase chain reaction (PCR) and direct sequencing. However, it is a time-consuming and complicated procedure, not suitable for routine clinical test. The objective of this study is to develop and evaluate a highly sensitive and rapid method using peptide nucleic acid (PNA) oligomers mediated real-time PCR clamping for detection of KRAS mutations. The PNA-mediated PCR clamping assay can real-time detect a mutation in a sample containing 1% of the mutant allele in a mixture of wild-type genomic DNA, which also enables the accurate and rapid detection of all KRAS codon 12 and 13 mutations in a single reaction. The total assay time is short as it requires only 1.5 hours after the samples preparation. Thus, the present method offers a potential alternative to be applied in clinical samples of CRC for detection of DNA carrying KRAS mutations.

Molecular testing of different cytologic preparations in patients with advanced lung adenocarcinoma: which yields the best results?

This study constitutes the first systematic comparison of molecular results between different cytology preparations in patients with lung adenocarcinoma undergoing testing for EGFR, KRAS, and BRAF mutations. 115 archival cytology preparations (direct smears, ThinPrep preparations [TP], and cell blocks [CB]) from lung adenocarcinomas with known EGFR, KRAS, or BRAF mutations were tested and compared with clinical testing results. Results were compared between preparations and analyzed in relation to tumor purity and tumor cell content. 82 (77%) of 106 informative cases were concordant with clinical testing results. There was no significant difference in the concordance rate between CB, TP, air-dried smears, or alcohol-fixed smears (P=0.3803), nor between preparations with <25%, 25% to 50%, or >50% tumor purity (P=0.1147). Concordance rates were lower in preparations with ≤100 tumor cells (P = 0.0002). Smears, TP, and CB are all valid substrates for molecular testing. Although tumor purity did not significantly affect results, low tumor content showed poorer performance. Recording tumor purity and content is recommended.

EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer(NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large‑scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism(SSCP)-Sanger sequencing and matrix‑assisted laser desorption ionization‑time of flight(MALDI‑TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9and98.1%, respectively, whereas the MALDI‑TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7and96.9%, respectively. The mean analytical turnaround times(TAT) were 4and3days for direct sequencing and the MALDI‑TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI‑TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time.