Role of circulating free DNA in evaluating clinical tumor burden and predicting survival in Chinese metastatic colorectal cancer patients.

BACKGROUND: The aim of this study was to explore the utility of circulating free DNA (cfDNA) in the evaluation of clinical tumor burden and survival in Chinese patients with metastatic colorectal cancer (mCRC) and to preliminarily summarize some metastatic characteristics associated with mutational status. METHODS: A panel covering a total of 197 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA was used to evaluate the mutational status in plasma by next-generation sequencing (NGS) technology in 126 patients with mCRC. An amplification-refractory mutation system (ARMS) was used to analyze genomic DNA from matched tissue samples. Clinical markers including carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), carbohydrate antigen 125 (CA125), neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) in serum and the sum of all tumor diameters on CT or PET/CT were collected to indicate clinical tumor burden. The correlations between cfDNA and clinical tumor burden were analyzed using Pearson correlation and linear regression models. The median progression-free survival (PFS) and 1-year overall survival (OS) rates were calculated by Kaplan-Meier (K-M) survival analysis. RESULTS: Of the 126 enrolled patients, patients who were tested positive for mutations in plasma accounted for 45.2% (57/126). Mutations in KRAS, NRAS, BRAF and PIK3CA were detected in 37.3% (47/126), 1.6% (2/126), 3.2% (4/126) and 13.5% (17/126) of patients, respectively. The overall concordance rate of mutational status between plasma and matched tissues was 78.6% (99/126). Sixteen patients had mutations in plasma that were not detected in tissue, including some rare hotspot mutations. The cfDNA concentration was significantly correlated with the levels of clinical markers, especially CEA (P < 0.0001, Pearson r = 0.81), LDH (P < 0.0001, Pearson r = 0.84) and the sum of tumor diameters (P < 0.0001, Pearson r = 0.80). Patients with a high cfDNA concentration (> 17.91 ng/ml) had shorter median progression-free survival (6.6 versus 11.7 months, P < 0.0001) and lower 1-year overall survival rate (56% versus 94%, P < 0.0001) than those with a low cfDNA concentration (≤17.91 ng/ml). The most common metastatic site was the liver (77.8%), followed by the lymph nodes (62.7%), lung (40.5%), peritoneum (14.3%) and bone (10.3%), in all patients. There was no significant difference in metastasis between different mutational statuses. CONCLUSION: Analyzing mutations in plasma could provide a more comprehensive overview of the mutational landscape than analyzing mutations in tissue. The cfDNA concentration could be a quantitative biomarker of tumor burden and could predict survival in Chinese patients with mCRC.

Analytical and clinical validation of a novel amplicon-based NGS assay for the evaluation of circulating tumor DNA in metastatic colorectal cancer patients.

Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

[One-stage total knee arthroplasty for femoral supracondylar fracture combined with knee osteoarthritis].

Objective: To evaluate the effectiveness of one-stage total knee arthroplasty (TKA) for femoral supracondylar fracture combined with knee osteoarthritis. Methods: Between January 2012 and March 2015, a total of 19 patients (19 knees) with femoral supracondylar fracture and knee osteoarthritis were treated with one-stage TKA. Of 19 cases, 8 were male and 11 were female with an average age of 69.6 years (range, 60-85 years). The mean body mass index was 22.6 kg/m 2 (range, 22.0-27.5 kg/m 2). The left knee was involved in 13 cases, and the right knee in 6 cases. The causes of femoral supracondylar fracture were falls in 10 cases, traffic accidents in 8 cases, and other injury in 1 case. All fractures were classified as type A according to AO/Association for the Study of Internal Fixation (AO/ASIF) classification. The interval of injury and operation was 4-13 days (mean, 8.6 days). The disease duration of osteoarthritis ranged from 30 to 90 months (mean, 52.6 months). During follow-up, the knee society score (KSS) and the range of motion (ROM) were used to evaluate the knee function; anteroposterior and lateral X-ray films of the knee were used to observe the position of the prosthesis. Results: All the incisions healed at the first stage, and there was no early complication such as pulmonary infection, pressure ulcer, and urinary tract infection. All patients were followed up 2-4 years with an average of 2.6 years. The ROM and KSS functional scores and clinical scores were significantly improved at 15 days and 2 years after operation, showing significant differences when compared with those before operation ( P<0.05). There were significant differences in the ROM and KSS functional scores and clinical scores between two time points after operation ( P<0.05). X-ray films showed the fracture bone healing, good alignment, no loosening of prosthesis at 2 years after operarion. Conclusion: One-stage TKA for femoral supracondylar fracture combined with knee osteoarthritis can achieve good effectiveness. It can not only reconstruct joint function, but also cure osteoarthritis and fracture at the same time, shorten the healing time, reduce the incidence of related complications.