Florent Mouliere

Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA

Assessment of KRAS status is mandatory in patients with metastatic colorectal cancer (mCRC) before applying targeted therapy. We describe here a blinded prospective study to compare KRAS and BRAF mutation status data obtained from the analysis of tumor tissue by routine gold-standard methods and of plasma DNA using a quantitative PCR–based method specifically designed to analyze circulating cell-free DNA (cfDNA). The mutation status was determined by both methods from 106 patient samples. cfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the seven tested KRAS point mutations, the method exhibited 98% specificity and 92% sensitivity with a concordance value of 96%. Mutation load, expressed as the proportion of mutant alleles in cfDNA, was highly variable (0.5–64.1%, median 10.5%) among mutated samples. CfDNA was detected in 100% of patients with mCRC. This study shows that liquid biopsy through cfDNA analysis could advantageously replace tumor-section analysis and expand the scope of personalized medicine for patients with cancer.

Liquid biopsies come of age: towards implementation of circulating tumour DNA

Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a liquid biopsy for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.

Circulating cell free DNA: Preanalytical considerations.

Despite the growing interest in circulating cell-free DNA (ccfDNA) analysis in various clinical fields, especially oncology and prenatal diagnosis, few studies on sample handling have been reported and no analytical consensus is available. The lack of consistency between the various protocols for sample handling and the techniques used for ccfDNA analysis is one of the major obstacles in translating ccfDNA analysis to clinical practice. Although this point is highlighted regularly in the published reviews on ccfDNA analysis, no standard operating procedure currently exists despite several ongoing clinical studies on ccfDNA analysis. This review examines the preanalytical parameters potentially affecting ccfDNA concentration and fragmentation at each preanalytical step from blood drawing to the storage of ccfDNA extracts. Analysis of data in the literature and our own observations revealed the influence of preanalytical factors on ccfDNA analysis. Based on these data, we determined the optimal preanalytical protocols for ccfDNA analysis and ultimately, a guideline for the translation of ccfDNA analysis in routine clinical practice.

High Fragmentation Characterizes Tumour-Derived Circulating DNA

Background Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments. Methodology In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients. Conclusion/Significance Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60–100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (nu200a=u200a12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (nu200a=u200a16).

Protective effect of BDNF against beta-amyloid induced neurotoxicity in vitro and in vivo in rats

We examined the potential protective effect of BDNF against beta-amyloid-induced neurotoxicity in vitro and in vivo in rats. In neuronal cultures, BDNF had specific and dose-response protective effects on neuronal toxicity induced by Abeta(1-42) and Abeta(25-35). It completely reversed the toxic action induced by Abeta(1-42) and partially that induced by Abeta(25-35). These effects involved TrkB receptor activation since they were inhibited by K252a. Catalytic BDNF receptors (TrkB.FL) were localized in vitro in cortical neurons (mRNA and protein). In in vivo experiments, Abeta(25-35) was administered into the indusium griseum or the third ventricle and several parameters were measured 7 days later to evaluate potential Abeta(25-35)/BDNF interactions, i.e. local measurement of BDNF release, number of hippocampal hilar cells expressing SRIH mRNA and assessment of the corpus callosum damage (morphological examination, pyknotic nuclei counting and axon labeling with anti-MBP antibody). We conclude that BDNF possesses neuroprotective properties against toxic effects of Abeta peptides.

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations.

Multi‐marker analysis of circulating cell‐free DNA toward personalized medicine for colorectal cancer

Development of a Q‐PCR‐based assay for the high‐performance analysis of circulating cell‐free DNA (ccfDNA) requires good knowledge of its structure and size.

Circulating tumor-derived DNA is shorter than somatic DNA in plasma

Circulating tumor DNA (ctDNA) is now widely investigated as a biomarker in translational and clinical research (1). However, despite the growing field of clinical applications, the biology of ctDNA remains unclear. In trying to learn about the origins of ctDNA, nature provides us with very few clues. One of the important accessible parameters is the size of those DNA fragments. In addition, a well-informed model of these sizes and biases can help design more efficient and accurate diagnostic methods. In PNAS, Jiang et al. take an important step in that direction (2).

Circulating DNA as a Strong Multimarker Prognostic Tool for Metastatic Colorectal Cancer Patient Management Care

Purpose: Circulating cell-free DNA (ccfDNA) is a valuable source of tumor material obtained from a simple blood sampling that enables noninvasive analysis of the tumor genome. Our goal was to carry out a multiparametric analysis of ccfDNA and evaluate its prognostic value by investigating the overall survival (OS) of 97 metastatic colorectal cancer patients (mCRC). Experimental Design: Qualitative parameters (determination of the main KRAS exon2 and BRAF V600E mutations) and quantitative parameters (total ccfDNA concentration, mutant ccfDNA concentration, the proportion of mutant ccfDNA, and ccfDNA integrity index) were determined simultaneously in a single run using a unique Q-PCR multimarker approach (100% success rate). Results: The median follow-up time was 36 months and median OS was 22 months. Patients showing high ccfDNA levels had significantly shorter OS (18.07 months vs. 28.5 months, P = 0.0087). Moreover, multivariate analysis revealed that a high ccfDNA level is an independent prognostic factor (P = 0.034). All ccfDNA parameters were of prognostic interest: patients with higher levels of mutant ccfDNA and higher mutation loads for the detected mutations had shorter OS (P = 0.0089 and P = 0.05, respectively). In addition, the level of ccfDNA fragmentation correlated positively with decreased OS in the exclusive KRAS/BRAF-mutant cohort of patients (P = 0.0052) and appeared as a strong independent prognostic factor (P = 0.0072), whereas it was not significant in the exclusive KRAS/BRAF WT cohort of patients (P = 0.67). Conclusions: Our data provide for the first time qualitative and quantitative evidence in favor of multiparametric ccfDNA analysis in mCRC patients for prognostic assessment. Clin Cancer Res; 22(12); 3067–77. ©2016 AACR.

The importance of examining the proportion of circulating DNA originating from tumor, microenvironment and normal cells in colorectal cancer patients

Introduction: The pressing need to determine the KRAS/BRAF mutational status for selecting patients with colorectal cancer (CRC) for anti-EGFR therapy provides a great opportunity to use circulating DNA (ctDNA) as a theranostic tool for personalized medicine. Better understanding of ctDNA origin (necrosis, apoptosis and active release) may increase the reliability of using abnormal ctDNA as biomarker. Areas covered: The authors showed that examining the proportion of ctDNA originating from tumor, microenvironment and normal cells, through size distribution and mutation load may help to discriminate mechanisms of ctDNA release. Expert opinion: Contrary to the literature, it was observed that tumor-derived ctDNA was mostly shorter than 100 bp. Tumor-derived ctDNA from cancer patients exhibited a specific ctDNA size distribution profile and significantly higher ctDNA fragmentation than ctDNA from healthy individuals. Examination of the KRAS and BRAF mutational load in 48 mutated samples revealed very high variation ranging from 0.037 to 68.8%. This suggests either that tumor cells variably release ctDNA compared with tumor-associated stroma cells or normal cells, or that mutant ctDNA analysis may depend on tumor clonality. Detection of point mutation by quantifying the proportion of mutant ctDNA fragments provides a powerful tool for assessing the proportion of ctDNA from different origins.