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Cell-free DNA (cfDNA) analysis, specifically circulating tumor DNA (ctDNA) analysis, provides enormous opportunities for noninvasive early assessment of cancers. To date, PCR-based methods have led this field. However, the limited sensitivity/specificity of PCR-based methods necessitates the search for new methods. Here, we describe a direct approach to detect KRAS G12D mutated genes in clinical ctDNA samples with the utmost LOD and sensitivity/specificity. In this study, MutS protein was immobilized on the tip of an atomic force microscope (AFM), and the protein sensed the mismatched sites of the duplex formed between the capture probe on the surface and mutated DNA. A noteworthy LOD (3 copies, 0.006% allele frequency) was achieved, along with superb sensitivity/specificity (100%/100%). These observations demonstrate that force-based AFM, in combination with the protein found in nature and properly designed capture probes/blockers, represents an exciting new avenue for ctDNA analysis.

A healthy 30-year-old man who had not had any prior medical or surgical history presented to the emergency department with substernal pain for 2 days that was sharp and exacerbated by inspiration and recumbency. Chest radiograph ( Figure 1A) showed mild left atrial enlargement. The ECG showed normal findings. The next day, the patient complained of sudden onset of class 3 dyspnea and frothy blood-tinged sputum. Follow-up chest radiography showed increased interstitial pulmonary vascular markings with consolidation in the right lower lung field, suggestive of pulmonary edema ( Figure 1B). Transthoracic echocardiogram showed minimal pericardial effusion and a cystlike, thin-walled, space-occupying lesion in the left atrium, which was not connected with the true lumen of the left atrium by color Doppler interrogation and contrast agent injection (Figure 2). Contrast cardiac computed tomography revealed a large homogenous mass in the posterior wall of the left atrium with a severely compressed left atrial chamber, which extended along the pulmonary veins, with tight luminal narrowing of the low pulmonary veins ( Figure 3). The patient underwent emergency surgery, and the condition was diagnosed as spontaneous left atrial dissection with minimal hemopericardium. From the Departments of Cardiology

Background Postoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay. Methods Personalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC). Results In 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49–20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001). Conclusion Tumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

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