Tumor DNA In Peripheral Blood Research Articles

Dynamic Changes of Circulating Tumor DNA in Peripheral Blood Predict the Efficacy of TKI in the Treatment of Lung Adenocarcinoma with EGFR Mutation

背景与目的有表皮生长因子受体（epidermal growth factor receptor, EGFR）突变的非小细胞肺腺癌，患者在给予酪氨酸激酶抑制剂（tyrosine kinase inhibitors, TKIs）的治疗中获得非常好的疗效，但绝大多数患者都会出现耐药，使得发现出现耐药的时间及可能耐药机制的检测有着越来越大的意义，目前二代基因测序方法（next generation sequencing, NGS）的出现使其成为可能。本文拟通过研究靶向治疗前后有EGFR突变的非小细胞肺癌循环肿瘤DNA（circulating tumor DNA, ctDNA）突变频率及突变谱的变化来监测靶向治疗效果。方法本中心入组22例通过组织活检或外周血ctDNA检查出EGFR突变的患者，分别于治疗前、服用TKI后2个月及临床进展时收集患者的外周血8 mL行ctDNA测序。结果EGFR敏感突变患者应用靶向药物治疗效果显著，EGFR敏感突变的患者在用TKI治疗后，相比治疗前突变基因突变丰度明显降低（P=0.015, 3）; 患者的中位无进展生存期较长（无进展生存时间=390 d）。同时我们发现伴随TP53基因突变时应用针对EGFR敏感突变的靶向药物治疗效果欠佳（中位无进展生存时间为120 d vs 630 d，P=0.000, 2）。结论EGFR敏感突变的患者在用TKI治疗后，突变基因突变丰度明显降低的患者的生存期更长（P < 0.05），突变丰度减低不明显或伴有其他突变者预示着TKI耐药。

High-performance electrochemical sensing of circulating tumor DNA in peripheral blood based on poly-xanthurenic acid functionalized MoS2 nanosheets

A high-performance sensing platform based on poly-xanthurenic acid (PXA) film functionalized MoS2 nanosheets was developed for electrochemical detection of circulating tumor DNA in peripheral blood. The MoS2 nanosheets were obtained using a simple ultrasonic method from bulk MoS2. The physical adsorption between MoS2 and aromatic XA monomers effectively improved the electropolymerization efficiency, accompanied with an increased electrochemical response of PXA. The obtained PXA/MoS2 nanocomposite not only served as a substrate for DNA immobilization but also reflected the electrochemical transduction originating from DNA immobilization and hybridization without any complex labelling processes or outer indicators. The immobilization of the probe ssDNA was achieved via noncovalent assembly due to the π–π interaction between PXA and DNA bases. After the hybridization of the probe ssDNA with the target DNA, the formation of helix structure induced the resulted dsDNA to be released from the surface of the PXA/MoS2 nanocomposite. The detection limit of this constructed DNA biosensor was calculated in the linear target DNA concentrations range from 1.0 × 10–16 mol/L to 1.0 × 10-10 mol/L and it was found to be 1.8 × 10–17 mol/L.

Systemic Therapy for Metastatic Colorectal Cancer: From Current Standards to Future Molecular Targeted Approaches.

Over the past 20 years, substantial advances have been made in the treatment of patients with metastatic colorectal cancer (mCRC). In particular, there is now a wide range of options for the front-line treatment of mCRC. Sophisticated molecular technologies have been developed to identify novel prognostic and predictive biomarkers for CRC. DNA sequencing technology has made remarkable advances in recent years, primarily as a result of the development of next-generation sequencing and whole exome sequencing, which are powerful new tools for the discovery of predictive molecular biomarkers to facilitate the delivery of personalized medicine. In addition to tumor tissue, recent efforts have focused on analyzing circulating tumor DNA in peripheral blood. Herein, we review the evolution of standard chemotherapy and targeted therapy strategies for the treatment of mCRC in the front-line setting, the molecular technologies that are presently being used to facilitate our ability to practice individualized medicine, and the practical aspects of applying molecular biomarkers to everyday clinical practice.

Systemic Therapy for Metastatic Colorectal Cancer: From Current Standards to Future Molecular Targeted Approaches

Over the past 20 years, substantial advances have been made in the treatment of patients with metastatic colorectal cancer (mCRC). In particular, there is now a wide range of options for the front-line treatment of mCRC. Sophisticated molecular technologies have been developed to identify novel prognostic and predictive biomarkers for CRC. DNA sequencing technology has made remarkable advances in recent years, primarily as a result of the development of next-generation sequencing and whole exome sequencing, which are powerful new tools for the discovery of predictive molecular biomarkers to facilitate the delivery of personalized medicine. In addition to tumor tissue, recent efforts have focused on analyzing circulating tumor DNA in peripheral blood. Herein, we review the evolution of standard chemotherapy and targeted therapy strategies for the treatment of mCRC in the front-line setting, the molecular technologies that are presently being used to facilitate our ability to practice individualized medicine, and the practical aspects of applying molecular biomarkers to everyday clinical practice.

Abstract 5233: Prediction and response evaluation of EGFR blockade for colorectal cancer by using circulating cell-free DNA

Abstract Aim: In 10% of metastatic colorectal cancer patients, discordance of KRAS mutational status between primary tumor and metastatic tumor is observed. However biopsy of liver or lung metastasis is invasive and risky. Liquid biopsy (LB) is a new method of molecular diagnosis using circulating tumor DNA in peripheral blood. Here, we evaluated KRAS mutations using LB as a biomarker to predict the efficacy of EGFR blockade and observed concentration variability of ccfDNA by chemotherapy. Methods: We enrolled 44 colorectal cancer patients (14 KRAS mutant and 30 KRAS wild in primary tumors) with distant metastases. Circulating cell-free DNA (ccfDNA) was purified from 1 mL serum using the QIAamp Circulating Nucleic Acid Kit. We quantitated the concentration of ccfDNA and detected nine KRAS (G12A, G12R, G12D, G12C, G12S, G12V, G13D, Q61H, and Q61R) mutations. We administered EGFR blockade to 13 patients with KRAS wild in their primary tumor as the first line chemotherapy, and used computed tomography 3 months after starting EGFR blockade therapy to evaluate its effect. The study protocol was approved by the Ethics Review Committee of our institution. Written informed consent was obtained from each patient. Results: KRAS mutations in ccfDNA were detected in 85% (12/14) of patients with KRAS mutation in their primary tumor but in 10% (3/30) of patients without KRAS mutations in their primary tumors. In one of the 3 patients, primary tumor had wild KRAS but metastatic liver tumor and ccfDNA was mutated KRAS. We administered EGFR blockade to 13 patients. Twelve patients with KRAS wild in ccfDNA had a good clinical response (1CR and 12PR). One patient with KRAS mutation (G13D) in ccfDNA had no response. In majority of metastatic colorectal cancer patients, concentration of ccfDNA is more than 1000ng/ml. After curative resection, concentration of ccfDNA is less than 100ng/ml in non-metastatic colorectal cancer patients. In chemotherapy effective cases, the concentration of ccfDNA decreased immediately after administration, but the concentration of ccfDNA did not decrease in non-effective case. In our CR case, ccfDNA did not disappear (minimum 180ng/ml) while CR was continuing and new metastatic liver tumor appeared 28 weeks after detection of ccfDNA marked increasing. Conclusions: LB detection of KRAS mutations is less-invasive and repeatable compared with current techniques and may be useful for prediction of EGFR blockade. The concentration of ccfDNA decreased immediately in objective response case that is administered EGFR blockade. Conversely, the concentration of ccfDNA increased before imaging detection of disease progression. Citation Format: Takuma Iwai, Takeshi Yamada, Hatato Kan, Michihiro Koizumi, Seiichi Shinji, Goro Takahashi, Atushi Watanabe, Satoshi Matumoto, Akihisa Matuda, Aya Yamagishi, Yasuyuki Yokoyama, Eiji Uchida. Prediction and response evaluation of EGFR blockade for colorectal cancer by using circulating cell-free DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2015-5233

243P - Liquid Biopsy Detection of Kras and Braf Mutations May Be Useful As a Prognostic or Predictive Marker

ABSTRACT Aim: Resistance to epidermal growth factor receptor (EGFR) blockade may occur by selection of pre-existing KRAS mutant clones or continuing mutagenesis caused by EGFR blockade. Serial biopsies to identify new mutations are invasive and risky, especially for cancers deep within the body. Liquid biopsy (LB) is a new method of molecular diagnosis using circulating tumor DNA in peripheral blood. Here, we evaluated KRAS and BRAF mutations using LB as a biomarker to predict the efficacy of EGFR blockade. Methods: We enrolled 29 colorectal cancer patients (14 KRAS mutant and 15 KRAS wild in primary tumors) with distant metastases. Circulating tumor DNA was purified from 1 mL serum using the QIAamp Circulating Nucleic Acid Kit. We detected nine KRAS (G12A, G12R, G12D, G12C, G12S, G12V, G13D, Q61H, and Q61R) and one BRAF (V600E) mutations. We administered EGFR blockade to 8 patients with KRAS wild in their primary tumor and used computed tomography 3 months after starting EGFR blockade therapy to evaluate its effect. The study protocol was approved by the Ethics Review Committee of our institution. Written informed consent was obtained from each patient. Results: KRAS mutations in circulating tumor DNA were detected in 86% of patients with KRAS mutation in their primary tumor but in 14% of patients without KRAS mutations in their primary tumors. We administered EGFR blockade to 12 patients. Nine patients with KRAS and BRAF wild in circulating tumor DNA had a good clinical response (1CR and 8PR). One patient with KRAS mutation (G13D) without BRAF mutation in circulating tumor DNA had partial response. Two patients with KRAS mutation (G13D and Q61R) without BRAF mutation in circulating tumor DNA had no clinical response. In a patient without KRAS or BRAF mutations in circulating tumor DNA, we detected KRAS mutation (G12C) in circulating tumor DNA 12 months after starting EGFR blockade. We detected radiographic evidence of disease progression 2 months after detecting KRAS mutation in circulating tumor DNA. Conclusions: LB detection of KRAS and BRAF mutations is less-invasive and repeatable compared with current techniques and may be useful for identifying predictive markers of EGFR blockade and resistance to EGFR blockade. Disclosure: All authors have declared no conflicts of interest.