DNA In Colorectal Cancer Patients Research Articles

Circulating Bacterial DNA in Colorectal Cancer Patients: The Potential Role of Fusobacterium nucleatum.

Intestinal dysbiosis is a major contributor to colorectal cancer (CRC) development, leading to bacterial translocation into the bloodstream. This study aimed to evaluate the presence of circulated bacterial DNA (cbDNA) in CRC patients (n = 75) and healthy individuals (n = 25). DNA extracted from peripheral blood was analyzed using PCR, with specific primers targeting 16S rRNA, Escherichia coli (E. coli), and Fusobacterium nucleatum (F. nucleatum). High 16S rRNA and E. coli detections were observed in all patients and controls. Only the detection of F. nucleatum was significantly higher in metastatic non-excised CRC, compared to controls (p < 0.001), non-metastatic excised CRC (p = 0.023), and metastatic excised CRC (p = 0.023). This effect was mainly attributed to the presence of the primary tumor (p = 0.006) but not the presence of distant metastases (p = 0.217). The association of cbDNA with other clinical parameters or co-morbidities was also evaluated, revealing a higher detection of E. coli in CRC patients with diabetes (p = 0.004). These results highlighted the importance of bacterial translocation in CRC patients and the potential role of F. nucleatum as an intratumoral oncomicrobe in CRC.

A new evaluation of the rearranged non-coding control region of JC virus in patients with colorectal cancer

HighlightsA significant proportion of 35/60 (58.33%) tumor tissue and 42/60 (70%) urine samples of the CRC patients were found to be positive for JCV DNA (P = 0.25). The parallel analysis of tumor and urine samples for JCV DNA further supports the potential for non-invasive screening tools.screening JCV DNA in peripheral blood mononuclear cells (PBMCs) and stool of CRC patients may further support the potential for non-invasive screening tools.The study offers new insights into rearranged NCCR variants isolated from colorectal cancer (CRC) patients’ tissue.The screening and analysis of the rrNCCR region of JCV DNA in CRC patients’ stool may indicate the pathogenesis of the JCV virus in CRC development.The presence of JCV LTAg in tumor tissue is significantly higher than in normal tissue (p = < 0.002), suggesting JCV’s role in cancer development during the latency phase.

&lt;i&gt;LINE-1&lt;/i&gt; hypomethylation and &lt;i&gt;HIST1H4F&lt;/i&gt; hypermethylation as oncomarkers in liquid biopsy of colorectal cancer

Introduction. Local hypermethylation of gene promoters and global genome hypomethylation are well-known manifestations of aberrant methylation associated with carcinogenesis. We investigated this phenomenon as a possible diagnostic marker for liquid biopsy of colorectal cancer using the original quantitative DNA melting analysis with hybridiza-tion probes (qDMA-HP) method. Aim. To quantify the methylation of HIST1H4F promoter and LINE-1 transposon in circulating blood plasma DNA of colorectal cancer patients. Materials and methods. Bisulfite-treated DNA samples isolated from blood plasma of healthy donors and cancer patients were analyzed. HIST1H4F methylation was assessed by asymmetric polymerase chain reaction with hybridized probe and post-amplification melting of probe / amplicon hybrids. To test for repetitive and highly polymorphic LINE-1 sequences, asymmetric polymerase chain reaction with hybridized probe and SYBR Green intercalating dye was used, followed by melting of hybrids and analysis of multicomponent melt curves. Results. High diagnostic efficiency of LINE-1 and HIST1H4F methylation markers in liquid biopsy of colorectal cancer was demonstrated with the area under the ROC curve = 0.92, sensitivity – 100 %, specificity – 84 %. Cross validation supports this result. Hypermethylation of HIST1H4F and hypomethylation of LINE-1 are statistically significantly correlated (Spearman correlation coefficient r = 0.4; p = 0.01). Conclusion. The qDMA-HP is suitable for quantitative assessment of aberrant methylation of various clinically significant genes.

The Prognostic Utility of KRAS Mutations in Tissue and Circulating Tumour DNA in Colorectal Cancer Patients

This study aims to investigate the long-term prognostic utility of circulating tumour DNA (ctDNA) KRAS mutations in colorectal cancer (CRC) patients and compare this with KRAS mutations in matched tissue samples. Tumour tissue (n = 107) and ctDNA (n = 80) were obtained from patients undergoing CRC resection and were analysed for KRAS mutations. The associations between KRAS mutation and overall survival (OS), cancer-specific survival (CSS), and recurrence-free survival (RFS) were analysed. All outcomes were measured in years (y). A total of 28.8% of patients had KRAS mutations in ctDNA and 72.9% in tumour tissue DNA. The high frequency of KRAS mutations in tissue samples was due to 51.4% of these being a detectable low mutation allele frequency (&lt;10% MAF). Comparing KRAS mutant (KRASmut) to KRAS wild-type (KRASwt) in ctDNA, there was no association found with OS (mean 4.67 y vs. 4.34 y, p = 0.832), CSS (mean 4.72 y vs. 4.49 y, p = 0.747), or RFS (mean 3.89 y vs. 4.26 y, p = 0.616). Similarly, comparing KRASmut to KRASwt in tissue DNA there was no association found with OS (mean 4.23 y vs. 4.61 y, p = 0.193), CSS (mean 4.41 y vs. 4.71 y, p = 0.312), or RFS (mean 4.16 y vs. 4.41 y, p = 0.443). There was no significant association found between KRAS mutations in either tissue or ctDNA and OS, CSS, or RFS.

Identıfıcatıon of KRAS Mutatıons in Colorectal Carcınoma Patıents at Dr. M. Djamıl Hospıtal, West Sumatra-Indonesıa

Kirsten rat sarcoma viral oncogene (KRAS) gene mutations lead to abnormal activation of the RAS signalling pathway and have been associated with poor prognosis and resistance to some therapeutics. This study aimed to identify mutation characteristics of the KRAS genes codon 12 and 13 in colorectal cancer patients in West Sumatra. KRAS mutations were analyzed in 20 DNA of colorectal cancer patients' tissue samples by using polymerase chain reaction (PCR) with specific primer and direct sequencing analysis. Our findings showed five samples (25%) with mutated KRAS at codons 12 and 13 (including three samples with GGT→GAT, one sample with GGT→GTT in codon 12, and one sample with GGC→GAC in codon 13). In conclusion, we found two variations of amino acid changes at codon 12 (G12D and G12V) and one at codon 13 (G13D). More research with many samples is required to obtain conclusive data on the relationship between these gene mutations and colorectal cancer response to therapy and prognosis.

Abstract 5715: Circulating tumor DNA in colorectal cancer patients with resectable liver metastases: Preliminary results of the MIRACLE study

Abstract Introduction: Recurrence risk after curative surgery for colorectal liver metastases (CRLM) remains high, underlining the need for novel prognostic markers. Liquid biopsies provide promising biomarkers to detect minimal residual disease (MRD) after local treatment. The aim of this study was to determine and compare the characteristics of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) in liquid biopsies taken before and after surgical treatment of CRLM. Materials &amp; Methods: Patients with isolated CRLM were recruited before undergoing potentially curative hepatic resection. Only patients who did not receive per-operative chemotherapy were selected. Peripheral blood samples were collected in CellSave preservative tubes at four different time points and processed into plasma within 96 hours after withdrawal. Subsequently, cfDNA was isolated from 4mL plasma, in which the total cfDNA concentration was measured. Mutations characterizing ctDNA were determined before surgery by next generation sequencing (NGS) using a colon-specific targeted panel (Oncomine Colon cfDNA assay, 14 genes). Samples were sequenced at &amp;gt;20.000 average reads depth and variants were called mutations when the variant allele frequency was above their predefined limit of detection. Longitudinal blood samples of ctDNA-positive patients were evaluated by digital PCR (dPCR) using mutation-specific assays, in which samples were considered ctDNA positive when &amp;gt;5 mutant copies were detected. Results: Blood samples of 231 patients were collected one day before surgery (T0), and were repeated one day (T1), one week (T2), and three weeks (T3) after surgery. The cfDNA yields per mL plasma were higher at T1 and T2 (median 93 ng and 64 ng, respectively) compared to T0 (6 ng, p&amp;lt;0.001) and T3 (12 ng, p&amp;lt;0.001). NGS at T0 was performed for 196 patients and detected ctDNA in 129 patients (66%). Mutations had a median allele frequency of 6.3% (ranging from 0.28 to 86.4%), and were observed in 12 genes, predominantly in TP53 (n=69), KRAS (n=57), APC (n=40), and PIK3CA (n=23). dPCR analyses were performed for 87 patients out of the 129 patients with ctDNA at T0, and confirmed TP53, KRAS, PIK3CA, and BRAF mutations in 75 patients, with average ctDNA loads of 9%, 8%, 18% and 4% for these genes. Longitudinal dPCR analyses were performed in 58 patients of whom subsequent post-surgical samples were available, and revealed that ctDNA was detectable in 22 patients (40%) at T1/T2, and in 21 patients (36%) at T3. Nine patients (16%) had detectable ctDNA at both T1/T2 and T3. Average mutation loads were 0.97% at T1/T2 and 1.79% at T3 (p=0.36). Discussion/Conclusion: ctDNA is a potential biomarker to detect MRD after curative treatment for CRLM. Our results demonstrate that out of all patients with detectable ctDNA at baseline (before surgery), ctDNA is still present in 36% of patients three weeks after resection. Citation Format: Lissa Wullaert, Maurice P. Jansen, Jaco Kraan, Jan M. van Rees, Corine M. Beaufort, Yannick M. Meyer, Boris Galjart, Pieter M. Nierop, Florian E. Buisman, Diederik J. Höppener, Erik P. van der Stok, Gianmarco Motta, Dirk J. Grünhagen, Henk M. Verheul, Stefan Sleijfer, Saskia M. Wilting, Cornelis Verhoef, John W. Martens. Circulating tumor DNA in colorectal cancer patients with resectable liver metastases: Preliminary results of the MIRACLE study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5715.

Investigation of Microbial Translocation, TLR and VDR Gene Polymorphisms, and Recurrence Risk in Stage III Colorectal Cancer Patients.

Simple SummaryGut microbial dysbiosis and microbial passage into the peripheral blood leads to colorectal cancer (CRC) and disease progression. Toll-like (TLR) and vitamin D (VDR) receptors play important role in the immune modulation and polymorphisms that may increase CRC risk. The aim of the current study was to demonstrate the prognostic value of microbial DNA fragments in the blood of stage III CRC patients and correlate such microbial detection to TLR/VDR polymorphisms. TLR/VDR polymorphisms and presence of microbial DNA in CRC patients highlight their role in cancer development and progression.Gut microbial dysbiosis and microbial passage into the peripheral blood leads to colorectal cancer (CRC) and disease progression. Toll-like (TLR) and vitamin D (VDR) receptors play important role in the immune modulation and polymorphisms that may increase CRC risk and death rates. The aim of the current study was to demonstrate the prognostic value of microbial DNA fragments in the blood of stage III CRC patients and correlate such microbial detection to TLR/VDR polymorphisms. Peripheral blood was collected from 132 patients for the detection of microbial DNA fragments, and TLR/VDR gene polymorphisms. In the detection of various microbial DNA fragments, TLR and VDR polymorphisms was significantly higher compared to healthy group. Homozygous individuals of either TLR or VDR polymorphisms had significantly higher detection rates of microbial DNA fragments. Mutational and MSI status were significantly correlated with TLR9 and VDR polymorphisms. Significantly shorter disease-free survival was associated with patients with BRAF mutated tumors and ApaI polymorphisms, whereas shorter overall survival was associated with the detection of C. albicans. The detection of B. fragilis, as demonstrated by the multivariate analysis, is an independent poor prognostic factor for shorter disease-free survival. TLR/VDR genetic variants were significantly correlated with the detection of microbial fragments in the blood, and this in turn is significantly associated with tumorigenesis and disease progression.

99P The differentially fragmented regions in cell-free DNA of colorectal cancer patients

Cell-free DNA analysis in cancer patients may reveal tumor-specific alterations, including mutations, aberrant methylation, and chromatin state changes. The latter are connected to shifts in transcription programs and may be considered as epigenetic markers of cancer. Several whole-genome sequencing studies have shown that the chromatin state of parental cells may be partially decoded from cfDNA fragmentation features. In this preliminary study, we aim to test an approach for identifying differentially fragmented regions in cell-free DNA of cancer patients.

Use of circulating tumor DNA in colorectal cancer patients to assess tumor burden and response to therapy: An observational study.

3528 Background: Residual disease after treatment for colorectal cancer (CRC) poses a risk for recurrence but imaging and CEA are limited in their capacity to detect residual disease. A simple test is needed for assessing treatment response. This study determined whether levels of methylated BCAT1/IKZF1 DNA in blood correlate with tumor burden and whether post-treatment levels inform efficacy of different treatments for CRC. Methods: Patients with primary CRC had blood collected prior to treatment (n = 282, 59.9% males, median age 68.5y). Cell free DNA (cfDNA) was extracted from plasma and assayed for methylation in BCAT1 and IKZF1. Detection of methylation in either gene deemed a sample positive; levels were expressed as %methylation (average methylation/average cfDNA). Positive patients had additional samples collected post-treatment for early stage CRC (surgery, n = 31), advanced/metastatic CRC (surgery + adjuvant chemotherapy, n = 15), and rectal cancer (neoadjuvant therapy, surgery +/- chemotherapy, n = 6), or following mid-therapy suspension of treatment in advanced CRC (n = 24). Tumor size was expressed as the maximum diameter of the primary (assessed surgically or by MRI). Results: Pretreatment results increased with CRC stage. Positivity by stage was: I, 23.7% (14/59); II, 62.1% (54/87); III, 68.6% (70/102); IV, 85.3% (29/34). Level by stage: I, 0.0%; II, 0.06%; III, 0.07%; IV, 4.07%, p &lt; 0.001). Pretreatment levels correlated significantly with tumor size (r = 0.372, p &lt; 0.001). Post-treatment blood was collected a median 2.4mo (IQR 1.7-3.9) after therapy completion. Positivity decreased after completing treatment (Table), with 88.4% of cases (46/52) becoming ctDNA negative. All cases with complete treatment had a reduction in biomarker levels, whereas in those with incomplete therapy, 54.5% (12/22) remained positive and the pre- and post-treatment levels were not significantly different. Of those positive after treatment, 13 had a further blood sample: 8 had become ctDNA negative and all but one remained disease free. Five remained positive and all had further suspected or confirmed disease. Conclusions: Levels of methylated BCAT1 and IKZF1 DNA in blood correlated with tumor burden; levels became undetectable in the majority of patients following completion of planned curative intent treatment. The methylated ctDNA blood test aids monitoring of responses to therapy and identification of those cases with residual cancer who might benefit from ongoing therapy.[Table: see text]

Practices and expectations on the use of circulating tumor DNA in colorectal cancer patients: A bi-national AGEO/AIOM/GERCOR/FFCD/FRENCH survey

BackgroundIncreasing evidence shows that circulating tumor DNA (ctDNA) is a valuable tool in providing molecular, prognostic, predictive and dynamic information in colorectal cancer (CRC) patients. This study aimed to make a picture of knowledge, practice, attitudes and expectations about ctDNA in CRC patients. Material and methodsAn online CRC-ctdna survey was distributed from November 2019 to January 2020 to French and Italian cooperative and scientific groups of Hepato-Gastroenterologists (HGE), Medical Oncologists (MO), Radiotherapists (RT) and Digestive Surgeons (DS). ResultsOverall, 307 physicians completed the survey (57% Italian; 43% French). Most of them were MO (62%) and HGE (24%). Affiliations were University Hospital (48%), Cancer Center (21%), General Hospital (21%) and Private Hospital (10%). Notably, half of respondents declared to have access to ctDNA in their daily practice. Of them, 53% uses ctDNA to assess RAS/BRAF status only, 46% for RAS/BRAF with other mutations and 1% only for other mutations. MO and HGE identified quick RAS profiling (P = 0.031) as the main reason of interest in the use of ctDNA. Physicians from University Hospitals and Cancer Centers prescribed more ctDNA (P < 0.001) and more often in house (P < 0.001). The main future expectations concerning ctDNA were to guide therapeutic strategies in metastatic (78%) and adjuvant (73%) settings, and to better/quicker profile disease at baseline (56%). ConclusionHalf of participants can order ctDNA in their daily practice. Molecular profiling of metastatic patients remains the main goal of ctDNA use and ctDNA-based therapeutic strategies are an expectation for the future in both adjuvant and metastatic settings.

Direct plasmonic detection of circulating RAS mutated DNA in colorectal cancer patients

RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 μL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy.

484P Practices and expectations on the use of circulating tumor DNA in colorectal cancer patients: A bi-national AGEO/AIOM/GERCOR/FFCD/FRENCH survey

Increasing evidence showed that circulating tumor DNA (ctDNA) could be a promising tool in providing molecular, prognostic, predictive and dynamic information in colorectal cancer (CRC) patients. However, it is still poorly and heterogeneously used in clinical practice. The present study aimed to provide a picture of knowledge, practice, attitudes and expectations about ctDNA in CRC.

Evaluation of a Liquid Biopsy Protocol using Ultra-Deep Massive Parallel Sequencing for Detecting and Quantifying Circulation Tumor DNA in Colorectal Cancer Patients

The identification and quantification of actionable mutations are critical for guiding targeted therapy and monitoring drug response in colorectal cancer. Liquid biopsy (LB) based on plasma cell-free DNA analysis has emerged as a noninvasive approach with many clinical advantages over conventional tissue sampling. Here, we developed a LB protocol using ultra-deep massive parallel sequencing and validated its clinical performance for detection and quantification of actionable mutations in three major driver genes (KRAS, NRAS and BRAF). The assay showed a 92% concordance for mutation detection between plasma and paired tissues and great reliability in quantification of variant allele frequency.

Detection of KRAS mutations in plasma cell-free DNA of colorectal cancer patients and comparison with cancer panel data for tissue samples of the same cancers.

Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell–free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.

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.

BMP3 promoter hypermethylation in plasma-derived cell-free DNA in colorectal cancer patients.

Detecting cfDNA in plasma or serum could serve as a 'liquid biopsy', for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.

Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. Methodology: The 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. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. 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). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression. 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, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [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 728. doi:10.1158/1538-7445.AM2017-728

Detection of PIK3CA Mutations in Plasma DNA of Colorectal Cancer Patients by an Ultra-Sensitive PNA-Mediated PCR.

Mutant Phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) has been shown to be associated with the occurrence, development and prognosis in colorectal cancer (CRC). However, its detection has been limited because of complicated procedures and the low sensitivity of the present approaches. We established an ultra-sensitive peptide nucleic acid-mediated polymerase chain reaction (PNA-PCR) assay to detect PIK3CA gene mutation in exon 9 and exon 20 with cell-free DNA (cfDNA). Using this technology, we detected the mutation status of PIK3CA in 128 colorectal cancer patients. 6 CRC patients receiving targeted therapy were chosen at random to undergo continuous PIK3CA mutation detection. The results showed that the sensitivity of PNA-PCR clamping method was 0.1% for the exon 9 and 0.2% for the exon 20 variant alleles. When the PIK3CA mutation status was determined by PNA-PCR plus sequencing, 38.3% (49/128) of CRC carried at least one mutation, either E545Kor H1047R. The clinic-pathological parameters of age (p=0.358), gender (p=0.622), disease stage (p=0.353) and disease location (p=0.307) were not associated with the PIK3CA mutation. In the continuous monitoring study, we found that the gene status was associated with the effect of treatment. Furthermore, when the PIK3CA variant was determined by only the PNA-PCR method, there was a good linear relationship between ΔCp values and the proportion of variant DNA. The accuracy of PNA-PCR was 93.75 and 92.27% respectively when the cut-off values of ΔCp at 9.0 and 8.0 were set for determining the E545K and H1047R mutations. A simple, noninvasive, ultra-sensitive PNA-PCR technology was developed and was especially suitable for the dynamic detection of PIK3CA variants using cfDNA.

Predictors of circulating tumor-derived methylated BCAT1 and IKZF1 DNA in colorectal cancer patients.

567 Background: Blood tests detecting circulating tumor-derived DNA (ctDNA) are being developed for colorectal cancer (CRC) screening and monitoring. However, the underlying tumor biology resulting in circulating DNA is not well understood. This study aimed to elucidate tumor features associated with methylated BCAT1 and IKZF1 DNA in blood as well as patient demographics that predict biomarker appearance. Methods: 129 people with invasive CRC had blood collected prior to surgery. Patient sociodemographic factors were recorded. Extracted circulating cell-free DNA was assayed for methylated BCAT1 and IKZF1. Patient factors and tumor features including location, size, depth of invasion (T stage), degree of differentiation, vascularity, lymphatic and perineurial invasion, lymph node involvement and presence of metastasis were compared with blood test results using multivariate logistic regression analysis. Results: The distribution of CRC stages I to IV were 34, 43, 41 and 11, respectively and 72 (56%) were blood positive for methylated BCAT1/IKZF1. Positivity was associated with distally located tumors and a higher T stage (Table). Gender, age, BMI, race, smoking and medications were not associated with blood positivity rates, but increased alcohol intake ( &gt; 7 drinks/week, n = 31) was associated (OR 5.7, 95% CI 1.1-28.7, p = 0.036). Conclusions: The strongest predictor for appearance of ctDNA was invasion depth of the tumor. Demographic features did not influence the positivity rate of the BCAT1/ IKZF1 CRC blood test, strengthening the validity of this blood test for CRC screening and monitoring in the general population. [Table: see text]

KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.