Mutation Detection Assay Research Articles

Effect of TXNRD2 rs35934224, FOXC1 rs2745599 and rs984253 genetic polymorphisms combinations on the development of primary open-angle glaucoma and their degree of association with the disease

Primary open-angle glaucoma (POAG) is a complex disease caused by numerous genetic and environmental factors, as well as their interaction. In recent studies, the effect of genetic polymorphisms combinations and non-equilibrium linkage of allele genes related to the development of POAG has been proved. The aim of the study was to determine the effect of TXNRD2 rs35934224, FOXC1 rs2745599 and rs984253 genetic polymorphisms combinations on the development of primary open-angle glaucoma and their degree of association with the disease. The study included 93 patients (185 eyes) with POAG stage I-IV and 89 volunteers (178 eyes) control subjects without any types of glaucoma. The patients were divided into four groups according to the degree of perimetric changes (Nesterov A. P., 2008). All patients performed visometry, computer perimetry, tonometry, biomicroscopy, ophthalmoscopy, gonioscopy, keratopahymetry, optical coherent tomography of the optic nerve. Analysis of the TXNRD2 rs35934224, FOXC1 rs2745599 and rs984253 genetic polymorphism with POAG was performed in real time using a polymerase chain reaction (PCR) in Gene Amp® PCR System 7500 (Applied Biosystems, USA) automatic amplifier. In the first stage of the study, the genomic DNA from whole venous blood was isolated using the standard reagents PureLink® Genomic DNA Kit for purification of genomic DNA, manufactured by INVITROGEN (USA). The analysis of polymorphism was carried out using unified test systems of TaqMan Mutation Detection Assays Life-Technology (USA). It was determined that the association with POAG had the genotype C/T*A/A*T/A as by comparing control with all patients, and by stratification – with the 1st, 2nd and 3rd groups of patients. The obtained results showed the evidentiary effect of this genotype combinations on the appearance of POAG, and on its progression by the stages of perimetric changes. The risk of the occurrence of POAG in carriers of genotypes C/T*A/A*T/A was increased by 2.8 times (p<0.001). In this combination, the two polymorphisms had heterozygous genotypes (rs35934224 – C/T, rs984253 – T/A), and the genotype rs2745599 – a mutant homozygote A/A. A combination of genotypes C/C*G/A*T/A was also important for the progression of the disease till stage II, which increased the risk of development of the POAG stage II by 2.9 times (p<0.01) compared to control. The risk of occurrence of the POAG in general and development of stage IV increased the presence of combinations of three minor genotypes T/T*A/A*A/A, which was encountered only in patients with POAG (in stage II – f = 0.025, in the third stage – f = 0.036, and in IV – f = 0.071). In our opinion, it confirmed the proposed working hypothesis of the study and showed that the more genotype combinations have the mutant alleles, the stronger this genotype affects the development of POAG.

Effect of Coexisting KRAS and TP53 Mutations in Patients Treated With Chemotherapy for Non–small-cell Lung Cancer

BackgroundKRAS and TP53 are common mutations in non–small-cell lung cancer (NSCLC). The Lung Adjuvant Cisplatin Evaluation Biological Program group found adjuvant chemotherapy to be deleterious in patients with coexisting KRAS/TP53 mutations. Patients and MethodsTo validate these results, patients with NSCLC tested for KRAS and TP53 mutations and receiving chemotherapy for any stage NSCLC were selected. Mutation status was analyzed using next generation sequencing (Illumina) or multiplex recurrent mutation detection (MassARRAY, Agena Biosciences) assays, and was correlated with clinical and demographic data. Disease-free (DFS) or progression-free survival (PFS) was the main endpoint, and overall survival (OS) was the secondary endpoint. ResultsAmong 218 patients, 28 had coexisting KRAS/TP53 mutations, 77 TP53, 37 KRAS, 76 had neither KRAS nor TP53 mutation (WT/WT). There was no DFS/PFS difference for the KRAS/TP53 group versus all others among 99 patients who received adjuvant chemotherapy (hazard ratio [HR], 1.22; 95% confidence interval [CI], 0.61-2.44; P = .57), 27 stage III patients who received chemo-radiation (HR, 0.87; 95% CI, 0.32-2.38; P = .8), and 63 patients who received palliative chemotherapy (HR, 0.68; 95% CI, 0.31-1.48; P = .33). OS was longer in the WT/WT group compared with any other group (KRAS: HR, 1.87; 95% CI, 1.02-3.43; P = .043; TP53: HR, 2.17; 95% CI, 1.3-3.61; P = .0028; KRAS/TP53: HR, 2.06; 95% CI, 1.09-3.88; P = .026). No OS difference was seen for KRAS/TP53 compared with the other groups (HR, 1.26; 95% CI, 0.75-2.13; P = .38). ConclusionsThere was no significant difference in DFS/PFS between the 4 groups. However, OS was longer for patients with TP53 and KRAS wild-type NSCLC who received chemotherapy for any stage compared with patients with KRAS, TP53 mutation, or double mutant tumors.

Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients.

Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarray-based assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5' domain, complementary to the target sequences and a 3' domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3’ reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy.

SIGNIFICANCE OF THE TUMOR NECROTIC FACTOR ALPHA IN DEVELOPMENT OF NEPHROPATHY IN TYPE 2 DIABETES MELLITUS

Relevance. The value of tumor necrosis factor alpha (TNFα) and the polymorphism of its gene rs1800629 for the development of type 2 diabetes mellitus (DM) has been shown in some studies but the mechanism of such an effect and role in some ethnic populations of patients is not fully understood.
 Objective: to find out the value of TNFα and polymorphism of its gene rs1800629 in the development of type 2 DM and its vascular complications.
 Materials and methods. The study involved data from 152 Ukrainian patients with type 2 DM, aged 34-80 years (53.9±8.4 years) and 95 healthy persons (control). According to the results of clinical and laboratory examinations, the presence of complications was determined and the stage of the disease was established. The blood level of TNFα was determined by the immuneenzyme method (Bender Medsystems, Austria); polymorphism rs1800629 – by real time polymerase chain reaction (TaqMan Mutation Detection Assays Life-Technology, USA). Statistical data processing was used by Statistica 10 (StatSoft, Inc., USA).
 Results. The blood level of TNFα in type 2 DM significantly increased in accordance with the severity of the disease (the maximum in the third stage – 7,1 times; p=3,2e-17), which influenced the development of retinopathy (β=0,012; p=0,049), nephropathy by glomerular filtration rate (β=0,011; p=0,007) and arterial hypertension (β=0,007; p=0,042); the maximum was the effect on the development of macroangiopathy of the lower extremities (β=0.033; p<0.001). Minor allele A rs1800629 increased (OR=1,71; 95% CI 1,11-2.65; p=0,015) risk of type 2 DM. For genotypes the connection with the disease is confirmed by the dominant model of inheritance (G/G versus G/A+A/A; OR=1,87; 95% CI 1,10-3,18; p=0,020). Allele A contributed to a decrease in the velocity of glomerular filtration and was associated with the development of nephropathy (χ2=6,38; p=0,041). This could be due to higher TNFα levels in G/A genotypes-carriers (1,2 times) and A/A (1,7 fold) compared to genotype G/G-carriers (p<0,001).
 Conclusion. The presence of the allele A rs1800629 was an important factor in the diabetic nephropathy development; one of the mechanisms of the vascular diabetic complications development was excessive expression of the TNFα gene, resulting in excessive synthesis of TNFα.

2296. Development of a Sequencing-Based Assay for Detection of CMV Antiviral Resistance Mutations to Letermovir in UL56

BackgroundAntiviral resistance to human cytomegalovirus (CMV) is a growing concern for immunocompromised patients on prolonged antiviral regimens, and CMV remains the most clinically significant infection following allogeneic hematopoietic-cell transplantation. Letermovir targets subunit 2 of the viral terminase complex (UL56) and is approved for CMV prophylaxis in adult stem cell transplant recipients. Resistance to letermovir is conferred by point mutations in the UL56 gene, and with the potential clinical need for antiviral resistance testing, we have developed a UL56 sequencing assay covering 23 identified resistance mutations. Here we summarize the performance characteristics of the UL56 antiviral resistance assay.MethodsThis assay uses automated nucleic acid extraction followed by CMV UL56-specific polymerase chain reaction (PCR). PCR products are subjected to cycle sequencing and capillary electrophoresis, and the resulting sequences are analyzed for the The presence of known resistance mutations between codons 229 and 369 of the UL56 gene. The assay’s limit of detection (LOD), precision and accuracy were validated in accordance with accepted regulatory standards using multiple laboratory and clinical CMV strains.ResultsThe LOD was determined to be 99 IU/mL. Precision was demonstrated with multiple sample replicates over three days of testing, with 100% amino acid concordance within the region of interest (ROI). The assay also accurately identified 100% of amino acids within the ROI of 30 unique CMV-positive de-identified clinical samples. While some polymorphisms were detected, no mutations conferring resistance were identified in the clinical samples tested, which is in agreement with the literature indicating that naturally occurring polymorphisms in the UL56 gene have not been shown to confer resistance to letermovir.ConclusionThe CMV UL56 antiviral resistance assay was shown to be a rapid and sensitive means of detecting mutations conferring letermovir resistance. This expands current CMV antiviral resistance testing, which includes UL54 and UL97 sequencing, and provides physicians with the ability to monitor for the emergence of antiviral resistance mutations to all current FDA-approved anti-CMV drugs.Disclosures J. Grantham, Viracor Eurofins Clinical Diagnostics: Employee, Salary. J. Nutt, Viracor Eurofins Clinical Diagnostics: Employee, Salary. A. Tyler, Viracor Eurofins Clinical Diagnostics: Employee, Salary. E. Bixler, Viracor Eurofins Clinical Diagnostics: Employee, Salary. M. Altrich, Viracor Eurofins Clinical Diagnostics: Employee, Salary. S. Kleiboeker, Viracor Eurofins Clinical Diagnostics: Employee, Salary.

Monitoring BRAF and NRAS mutations with cell-free circulating tumor DNA from metastatic melanoma patients.

The mutation status of the BRAF and NRAS genes in tumor tissue is used to select patients with metastatic melanoma for targeted therapy. Cell-free circulating DNA (cfDNA) represents an accessible, non-invasive surrogate sample that could provide a snapshot of the BRAF and NRAS genotype in these patients.We investigated the feasibility of the Idylla™ assay for detection of BRAF and NRAS mutations in cfDNA of 19 patients with metastatic melanoma at baseline and during the course of treatment. The cfDNA genotype obtained with Idylla was compared to the results obtained with matched-tumor tissue and to clinical outcome.At baseline, 47% of patients harbored a BRAFV600 mutation in their cfDNA. Two months after targeted treatment the BRAFV600 mutant cfDNA was undetectable in all patients and 3 were disease-free. Moreover, 15% of patients harbored a NRAS mutation that was detected with plasma before treatment. The sensitivity and specificity were 80% and 89% for the BRAF status, and 79% and 100% for the NRAS status in pretreatment cfDNA compared to results obtained with a tissue test. Due to the small size of the population, no significant correlation was observed between the presence of BRAF or NRAS mutations in cfDNA and the metastatic tumor load or overall survival.In conclusion, this study demonstrated that evaluation with the Idylla system of the BRAF and NRAS mutation status in cfDNA may be a surrogate for determination of the BRAF and NRAS status in tumor tissue.

Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.

T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency.

We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selectivewild-type suppression by the T-blocker concept enabled 0.1% detection sensitivity using the intercalating dye-based qPCR chemistry instead of more complex target-specific dye-labeled probes. Excellent consistency in sensitivity and specificity of the T-blocker assay concept was demonstrated.

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.

A digital PCR assay development to detect EGFR T790M mutation in NSCLC patients

Epidermal growth factor receptor (EGFR) is a crucial factor in the development of non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitor (TKI) therapy requires a sensitive and accurate assay for detection of drug resistant mutations in patients undergoing TKI-based targeted therapy. Digital polymerase chain reaction (PCR) is a highly sensitive PCR based detection method with the capability of absolute quantification. Here we present a digital PCR based liquid biopsy for EGFR T790M detection from blood samples with a low detection limit of 0.1% mutant allele frequency. The assay shows 100% concordance with test results obtained from cFDA approved ARMS PCR based EGFR mutation detection assay on FFPE tissue sections. It also demonstrated satisfactory sensitivity and specificity at 62.5% and 100% respectively. This assay can satisfy the clinical need of detecting EGFR T790M mutations at extremely low allele frequency. It can be used as both a companion diagnostic tool and a monitoring strategy for the development of drug resistance, and to evaluate therapeutic effects on disease progression.

A Highly Verified Assay for KRAS Mutation Detection in Tissue and Plasma of Lung, Colorectal, and Pancreatic Cancer.

KRAS Mutation Test v2 is used for the qualitative detection and identification of 28 mutations in exons 2, 3, and 4 of the human KRAS gene. To verify the performance of KRAS Mutation Test v2 and to evaluate its accuracy by correlation with a next-generation sequencing method on Illumina MiSeq. In this study, we used formalin-fixed, paraffin-embedded tissue and plasma specimens from non-small cell lung cancer, colorectal cancer, and pancreatic cancer patients. Results of specificity, precision, analytical sensitivity, and accuracy as compared with a MiSeq method are reported. The KRAS Mutation Test v2 demonstrated exquisite sensitivity and specificity and broad coverage of KRAS mutations. Precision was 100% (108 of 108) across all samples, operators, and instruments for formalin-fixed, paraffin-embedded tissue and 99.8% (615 of 616) for plasma. Analytical sensitivity was high with detection of 1% mutant sequence in formalin-fixed, paraffin-embedded tissue samples and as low as 25 mutant sequence copies/mL for plasma samples. The test also showed high overall concordance for formalin-fixed, paraffin-embedded tumor tissue as well as for plasma specimens when compared with MiSeq sequencing results. The KRAS Mutation Test v2 is a highly robust, reproducible, and sensitive test for the qualitative detection of 28 mutations in exons 2, 3, and 4 of the KRAS gene in both solid (tissue) and liquid (plasma) biopsies from colorectal cancer, non-small cell lung cancer, and pancreatic cancer, and is a convenient option for KRAS mutation testing.

Evaluation of a fluorescence resonance energy transfer quantitative polymerase chain reaction assay for identification of gyrA mutations conferring enrofloxacin resistance in canine urinary Escherichia coli isolates and canine urine specimens.

OBJECTIVE To evaluate a fluorescence resonance energy transfer quantitative PCR (FRET-qPCR) assay for detection of gyrA mutations conferring fluoroquinolone resistance in canine urinary Escherichia coli isolates and canine urine specimens. SAMPLE 264 canine urinary E coli isolates and 283 clinical canine urine specimens. PROCEDURES The E coli isolates were used to validate the FRET-qPCR assay. Urine specimens were evaluated by bacterial culture and identification, isolate enrofloxacin susceptibility testing, and FRET-qPCR assay. Sensitivity and specificity of the FRET-qPCR assay for detection of gyrA mutations in urine specimens and in E coli isolated from urine specimens were computed, with results of enrofloxacin susceptibility testing used as the reference standard. RESULTS The validated FRET-qPCR assay discriminated between enrofloxacin-resistant and enrofloxacin-susceptible E coli isolates with an area under the receiver operating characteristic curve of 0.92. The assay accurately identified 25 of 40 urine specimens as containing enrofloxacin-resistant isolates (sensitivity, 62.5%) and 226 of 243 urine specimens as containing enrofloxacin-susceptible isolates (specificity, 93.0%). When the same assay was performed on E coli isolates recovered from these specimens, sensitivity (77.8%) and specificity (94.8%) increased. Moderate agreement was achieved between results of the FRET-qPCR assay and enrofloxacin susceptibility testing for E coli isolates recovered from urine specimens. CONCLUSIONS AND CLINICAL RELEVANCE The FRET-qPCR assay was able to rapidly distinguish between enrofloxacin-resistant and enrofloxacin-susceptible E coli in canine clinical urine specimens through detection of gyrA mutations. Therefore, the assay may be useful in clinical settings to screen such specimens for enrofloxacin-resistant E coli to avoid inappropriate use of enrofloxacin and contributing to antimicrobial resistance.

Abstract 2225: Limitations on mutation detection for early detection of cancer

Abstract Introduction: Cell-free DNA (cfDNA) has potential utility for early non-invasive detection of cancer. We assess the feasibility of cfDNA mutational assays for early detection based on their physiological and economic requirements. We further review alternative biological signals of early cancer and the potential of machine learning to integrate these signals into reliable diagnostics. Methods: A binomial model was used to assess depth and input requirements, with parameters derived from published data on cfDNA sequencing. Alternative strategies for early detection were assessed by literature review. Results: 30,000x unique coverage is required for 95% sensitivity for 1 mutant read at 0.01% variant allele frequency, requiring 180ng cfDNA input at 50% process efficiency; current sequencing costs and reimbursement levels may make such tests economically infeasible (Table). 5th percentile plasma cfDNA concentration in the screening population is ~2.3 ng/mL, requiring >140mL blood collection for test failure rate <5%. Somatic mutational heterogeneity creates significant specificity challenges. Proteins, RNAs, and compartmentalized macromolecules are more abundant and may overcome cfDNA's issues of quantity, but irrelevant biological processes may confound the data. New techniques in machine learning, including latent variable modeling, may be used to integrate heterogeneous data from multiple sources to overcome these challenges. Conclusions: Mutation-detection assays may not be feasible for early cancer detection due to limited sensitivity arising from low concentrations of cfDNA, heterogeneity leading to specificity challenges, and prohibitive cost. Integrating markers beyond mutations with modern machine learning may provide a potential route to statistically robust biomarker development for early cancer detection. Table. Assay requirements for tumor liquid biopsy and mutation-based early cancer detection. Sequencing cost by panel sizeVAF 95% sensitivityCorrected depthRaw depthInput volume (blood)TEC-Seq1 58 genes 81 kbRazavi et al2 508 genes 2000 KbWES ~20k genes 50,000 KbTumor liquid biopsy0.1%3000x15,000x15 mL$14$340$8300Early cancer detection0.01%30,000x150,000x150 mL$140$3400$83,0001Phallen J, et al. Sci Transl Med. 2017 Aug 16;9(403). 2Razavi P, et al. J Clin Oncol. 2017;35(suppl):abstr 11526.Model parameters: 1) No more than 5% of samples may fail because of insufficient cfDNA quantity; 2) 95% sensitivity to detect one read from any cancer-derived allele, assuming that one is present in the sample; 3) 50% process efficiency: half of the cfDNA molecules in the input blood sample are represented in the sequencer output; 4) 5x oversampling in sequencing for error correction; 5) 100% on-target rate in target enrichment; 6) “$1000 genome” sequencing costs: US $1000/(30 x 3 Gbp) of sequencing bandwidth; 7) only sequencing costs computed; all other costs (labor, equipment, facilities, depreciation, etc.) accounted at $0; 8) panel expansion neither reduces input requirements nor increases sequencing requirements.VAF = variant allele frequency; TEC-Seq = targeted error correction sequencing; WES = whole exome sequencing. Citation Format: Imran S. Haque, Gabriel Otte, Olivier Elemento. Limitations on mutation detection for early detection of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2225.

Abstract 939: Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients

Abstract Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA. The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots. The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965). This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients. Citation Format: Samuel Woodhouse, Vincent Plagnol, Karen Howarth, Stefanie Lensing, Matt Smith, Michael Epstein, Mikidache Madi, Sarah Smalley, Catherine Leroy, Jonathan Hinton, Frank de Kievit, Esther Musgrave-Brown, Colin Herd, Katherine Baker-Neblett, Will Brennan, Peter Dimitrov, Nathan Campbell, Nitzan Rosenfeld, James Clark, Davina Gale, Jamie Platt, John Calaway, Greg Jones, Tim Forshew. Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 939.

Abstract 3900: Plasma ctDNA multigene mutation analysis for the diagnosis and treatment monitoring of colorectal cancer patients

Abstract Introduction Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Also during therapy colorectal tumors can develop resistance due to mutations in cellular growth factor receptor signaling pathway genes such as KRAS and BRAF. Herein we report new validation studies of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated mutations including the tumor suppressor APC, growth factor signaling pathway genes KRAS and BRAF and the essential WNT pathway signaling gene Beta- catenin (CTNNB1) called ColoScapeTM .The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. While tumor-tissue remains the ‘gold standard' for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples; so called 'liquid biopsy'. Here we broaden our previous study on the initial validation of FFPE samples to include plasma DNA in colorectal cancer patients and by evaluating its clinical utility as a method for monitoring tumor ctDNA mutations during and after therapy as well as a potential screening test for early diagnosis of colorectal cancer. Methodology he high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP's) and insertion/deletions (indels) mutations can be detected. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). This study presents further validation of the test as a potential landmark clinical test for colorectal cancer initial detection in FIT posiive patients and for recurrence and therapy monitoring Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in Colon Cancer development, early detection, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Qing Sun, Jinwei Du, Larry Pastor, Jyoti Phatak, Jenny Wilding, Walter F. Bodmer, Aiguo Zhang. Plasma ctDNA multigene mutation analysis for the diagnosis and treatment monitoring of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3900.

Abstract 3251: TaqMan Advanced miRNA cDNA Synthesis Kit to simultaneously study expression of miRNA and mRNA and to detect somatic mutations by qPCR

Abstract MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. (For research use only. Not for use in diagnostic purposes.) Citation Format: Kathleen Hayashibara, Harita Veereshlingam, Malte Buchholz. TaqMan Advanced miRNA cDNA Synthesis Kit to simultaneously study expression of miRNA and mRNA and to detect somatic mutations by qPCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3251.

Abstract 5577: Single cell mutation and gene expression co-analysis of lung circulating tumor cells

Abstract With the aim of personalized medicine, there have been many targeted therapies developed against cancer-specific antigens. The efficacy of targeted therapies was first shown in lung cancer patients containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors. Despite the promise of these EGFR targeted therapies, lung cancer patients routinely acquire an additional EGFR mutation, T790M, which causes treatment resistance. In order to determine if a patient qualifies for targeted therapy, they must get an invasive tumor biopsy, and may require a second biopsy if they stop responding to treatment. Circulating tumor cells (CTCs) are shed from the primary tumor into the bloodstream, a fraction of which lead to the development of metastases. CTCs can be isolated from a minimally-invasive blood draw, termed a “liquid-biopsy”, and their increased presence in the blood correlates with poor patient prognosis. CTCs present an approach to track and screen patients during treatment to determine if they acquire new mutations or other aggressive cell phenotypes. CTCs have been shown to be highly heterogeneous, existing in phenotypic states across the entire epithelial to mesenchymal transition spectrum as well as contain both primary tumor matched and unmatched mutations. We have previously established a workflow for the isolation and single cell characterization of CTCs. CTCs are isolated from the blood using a novel high-throughput inertial microfluidic device, the Labyrinth. This device has been optimized using cancer cell lines across many cancer types and has been used to process hundreds of blood samples from breast, lung, and pancreatic cancer patients for CTC enumeration. After CTC isolation using the Labyrinth, single cell suspensions are generated for lysis and RT-qPCR for up to 96 single cells using a 96-gene panel. Here we present initial results for the incorporation of mutation screening on the same single cells using digital PCR (dPCR). dPCR relies on small sample quantity to load one target molecule per reaction droplet for end-point PCR analysis. Due to its single molecule resolution, it has increased sensitivity compared to other traditional PCR methods, making it an ideal platform for single cell mutation screening. This enables us to determine if the cell is homo- or heterozygous, as well as quantify the relative expression in heterozygous mutations. We have validated the technology for single cell mutation detection of EGFR mutations, L858R and T790M, in addition to other genes such as TP53 and KRAS. The specificity of the EGFR mutation detection assays was confirmed using lung cancer cell line H1975, which is known to carry these two heterozygous mutations. Ultimately, the ability to couple single cell mutation and gene expression analysis using CTCs may allow oncologists to better monitor treatment response to guide the use and duration of targeted therapies. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Sunitha Nagrath. Single cell mutation and gene expression co-analysis of lung circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5577.

Abstract LB-221: Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer

Abstract Purpose: To demonstrate that an early increase in ctDNA plasmatic levels after 14 days may identify patients (pts) with advanced chemorefractory colorectal cancer (aCRC) unlikely to benefit from regorafenib. Experimental procedures: The RegARd-C prospective multicentric clinical trial (NCT01929616) collected archival tumor tissue and plasma samples at baseline (BL), 14 days (D14) and every 2 months after treatment initiation in aCRC pts (n=141). A targeted gene sequencing was performed on pts' tumor tissue to identify tumor-specific mutations using a panel of 47 genes commonly mutated in CRC. To monitor circulating tumor DNA (ctDNA) levels in plasma samples collected at BL and D14 via droplet digital PCR (Bio-Rad QX200 ddPCR system), we selected tumor-specific mutations based on their allelic frequency. Results: As foreseen, in 96 pts the most common mutated genes were APC (73%), TP53 (72%), KRAS (66%), and PI3KCA (23%). Among these pts, 24 (25%), 49 (51%), 18 (19%) and 5 (5%) had respectively one, two, three and four tumor-specific mutations followed via ddPCR. On average 2 (range 1-4) tumor-specific mutations were followed per patient. In total, 65 specific paired PrimePCRTM ddPCRTM Mutation Detection Assays were optimized following digital MIQE guidelines to assess ctDNA levels. As previously described1, ctDNA levels were assessed in terms of absolute values of mutated copies per mL of plasma to avoid bias in the mutated/mutated + wild-type copies ratio. We considered an increase in ctDNA level of at least 12% as significant2. Pts were separated according to the presence (n=53) or absence (n=43) of at least one increasing mutation between BL and D14. An increase of at least one tumor-specific mutation as compared to none is associated with a significantly worse clinical outcome with a median PFS of 1.3 months versus 3.0 months (HR 1.88, P=0.002, 95% CI 1.24-2.85) and a median OS of 3.9 months versus 8.5 months (HR 2.04, P<0.001, 95% CI 1.33-3.12). Conclusion: Early (14 days) change in ctDNA levels under regorafenib therapy describes adequately the outcome of patients and may be used as a reliable tool for treatment personalization. Additionally, we confirmed the importance of using the absolute value of ctDNA instead of the mutated/total copies ratio usually reported in the literature for monitoring the outcome of a toxic treatment such as regorafenib. 1Kehagias P. et al. Oral presentation. LKI symposium, 2017; Leuven 2Whale AS. et al. Anal Chem. 2017 Citation Format: Pashalina Kehagias, Caroline Vandeputte, Lieveke Ameye, Hakim El Housni, Jean-François Laes, Amélie Deleporte, Karen Geboes, Thierry Delaunoit, Gauthier Demolin, Marc Peeters, Lionel D'Hondt, Jos Janssens, Javier Carrasco, Maria Gomez Galdon, Ghanem Ghanem, Pierre Heimann, Marianne Paesmans, Patrick Flamen, Alain Hendlisz. Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-221.

Abstract 5531: Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAF mutant metastatic melanoma undergoing treatment with BRAF-targeted therapies

Abstract Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies. Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw. Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget. Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge. Citation Format: Broderick Corless, Greg Chang, Weihua Liu, Jin Li, Andre Marziali, Laura Mai, Matthew Wiggin, Melissa Wilson, Anna Pavlick, Iman Osman, George Karlin-Neumann, Cindy Spittle, David Polsky. Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAFmutant metastatic melanoma undergoing treatment with BRAF-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5531.

Hotspot mutations in cancer genes may be missed in routine diagnostics due to neighbouring sequence variants

The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing.We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented.