An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

Complementing Tissue Testing With Plasma Mutation Profiling Improves Therapeutic Decision-Making for Patients With Lung Cancer.

BACKGROUND: Tissue biopsy is an integral part of the diagnostic approach to lung cancer. It is however invasive and limited by heterogeneity. Liquid biopsies may complement tissue testing by providing additional molecular information and may be particularly helpful in patients from whom obtaining sufficient tissue for genomic profiling is challenging. METHODS: Patients with suspected lung cancer (n = 71) were prospectively recruited. Blood and diagnostic tissue samples were collected within 48 h of each other. Plasma cell-free DNA (cfDNA) testing was done using an ultrasensitive amplicon-based next-generation sequencing (NGS) panel (plasma NGS testing). For cases diagnosed as non-small cell lung carcinoma (NSCLC) via histology or cytology, targeted testing for epidermal growth factor receptor (EGFR) mutations was performed using tissue biopsy samples (tissue EGFR testing), where available. Concordance of clinically actionable mutations between methods and sample types was assessed. RESULTS: For confirmed NSCLC cases (n = 54), tissue EGFR test results were available only for 70.3% (38/54) due to sample inadequacies, compared to blood samples for 98.1% (53/54) cases. Tissue EGFR testing identified sensitizing EGFR (L858R or exon 19 deletion) mutation in 31.6% (12/38) of cases. Plasma NGS identified clinically actionable mutations in 37.7% (20/53) of cases, including EGFR mutations in two cases with no tissue EGFR results, and mutations in KRAS, BRAF, and MET. The overall sensitivity of sensitizing EGFR mutation detection by plasma NGS was 75% (9/12), and specificity was 100% (25/25) in patients tested in both tissue EGFR and plasma NGS (n = 37). In this cohort of patients, tissue EGFR testing alone informed clinical decisions in 22.2% (12/54) of cases. Adding plasma NGS to tissue EGFR testing increased the detection rate of actionable mutations to 42.6% (23/54), representing a 1.9-fold increase in clinically relevant findings. The average turnaround time of plasma NGS was shorter than standard tissue testing (10 vs. 29.9 days, p < 0.05). CONCLUSIONS: In the first-line setting, plasma NGS was highly concordant with tissue EGFR testing. Plasma NGS increases the detection of actionable findings with a shorter time to results. This study outlines the clinical utility of complementary plasma mutation profiling in the routine management of lung cancer patients.

Neoadjuvant chemotherapy plus intensity-modulated radiotherapy versus concurrent chemoradiotherapy plus adjuvant chemotherapy for the treatment of locoregionally advanced nasopharyngeal carcinoma: a retrospective controlled study.

BACKGROUND: In the era of intensity-modulated radiotherapy (IMRT), the role of neoadjuvant chemotherapy (NAC) for locoregionally advanced nasopharyngeal carcinoma (NPC) is under-evaluated. The aim of this study was to compare the efficacy of NAC plus IMRT and concurrent chemoradiotherapy (CCRT) plus adjuvant chemotherapy (AC) on locoregionally advanced NPC. METHODS: Between January 2004 and December 2008, 240 cases of locoregionally advanced NPC confirmed by pathologic assessment in Sun Yat-sen University Cancer Center were reviewed. Of the 240 patients, 117 received NAC followed by IMRT, and 123 were treated with CCRT plus AC. The NAC + IMRT group received a regimen that included cisplatin and 5-fluorouracil (5-FU). The CCRT + AC group received cisplatin concurrently with radiotherapy, and subsequently received adjuvant cisplatin and 5-FU. The survival rates were assessed by Kaplan-Meier analysis, and the survival curves were compared using a log-rank test. Multivariate analysis was conducted using the Cox proportional hazard regression model. RESULTS: The 5-year overall survival (OS), locoregional relapse-free survival (LRRFS), distant metastasis-free survival (DMFS), and disease-free survival (DFS) were 78.0, 87.9, 79.0, and 69.8%, respectively, for the NAC + IMRT group and 78.7, 84.8, 76.2, and 65.6%, respectively, for the CCRT + AC group. There were no significant differences in survival between the two groups. In multivariate analysis, age (<50 years vs. ≥50 years) and overall stage (III vs. IV) were found to be independent predictors for OS and DFS; furthermore, the overall stage was a significant prognostic factor for DMFS. Compared with the CCRT + AC protocol, the NAC + IMRT protocol significantly reduced the occurrence rates of grade 3-4 nausea-vomiting (6.5 vs. 1.5%, P = 0.023) and leukopenia (9.7 vs. 0.8%, P = 0.006). CONCLUSIONS: The treatment outcomes of the NAC + IMRT and CCRT + AC groups were similar. Distant metastasis remained the predominant mode of treatment failure.

A model to predict the risk of lethal nasopharyngeal necrosis after re-irradiation with intensity-modulated radiotherapy in nasopharyngeal carcinoma patients.

BACKGROUND: For patients with nasopharyngeal carcinoma (NPC) who undergo re-irradiation with intensity-modulated radiotherapy (IMRT), lethal nasopharyngeal necrosis (LNN) is a severe late adverse event. The purpose of this study was to identify risk factors for LNN and develop a model to predict LNN after radical re-irradiation with IMRT in patients with recurrent NPC. METHODS: Patients who underwent radical re-irradiation with IMRT for locally recurrent NPC between March 2001 and December 2011 and who had no evidence of distant metastasis were included in this study. Clinical characteristics, including recurrent carcinoma conditions and dosimetric features, were evaluated as candidate risk factors for LNN. Logistic regression analysis was used to identify independent risk factors and construct the predictive scoring model. RESULTS: Among 228 patients enrolled in this study, 204 were at risk of developing LNN based on risk analysis. Of the 204 patients treated, 31 (15.2%) developed LNN. Logistic regression analysis showed that female sex (P = 0.008), necrosis before re-irradiation (P = 0.008), accumulated total prescription dose to the gross tumor volume (GTV) ≥145.5 Gy (P = 0.043), and recurrent tumor volume ≥25.38 cm(3) (P = 0.009) were independent risk factors for LNN. A model to predict LNN was then constructed that included these four independent risk factors. CONCLUSIONS: A model that includes sex, necrosis before re-irradiation, accumulated total prescription dose to GTV, and recurrent tumor volume can effectively predict the risk of developing LNN in NPC patients who undergo radical re-irradiation with IMRT.

A prognostic model predicts the risk of distant metastasis and death for patients with nasopharyngeal carcinoma based on pre-treatment serum C-reactive protein and N-classification.

PURPOSE: Chronic inflammation plays an important role in nasopharyngeal carcinoma (NPC) development and progression. Aim of this study is to determine whether inflammation-related parameters predict distant metastasis in NPC patients. MATERIALS AND METHODS: 335 newly diagnosed non-metastatic NPC patients were recruited. The values of the C-reactive protein (CRP), lactate dehydrogenase, albumin, globulin, white blood cell and neutrophil at baseline were measured. RESULTS: Among the above six parameters, only CRP was independently associated with distant metastasis-free survival (DMFS). CRP concentration of advanced T-/TNM-classification patients was higher than those with early classification (P = 0.001). Higher-CRP (CRP ⩾ 2.46 mg/L) predicted shorter overall survival, disease-free survival and DMFS than lower-CRP (CRP < 2.46 mg/L). In a multivariable model, higher-CRP and advanced N-classification were independent predictors of distant metastasis. On the basis of these two parameters, a prognostic NC-model was developed as following: (1) low-risk (early N-classification and lower-CRP); (2) intermediate-risk (advanced N-classification or higher-CRP) and (3) the high-risk distant metastasis (advanced N-classification and higher-CRP). When compared with the low-risk group, the hazard ratios (HRs) for distant metastasis and death for the intermediate-/high-risk patients were 3.6/16.1 and 2.26/7.61, respectively (both P < 0.001). CONCLUSION: We developed a new prognostic model based on CRP and N-classification for predicting distant metastasis and death of NPC patients, which may facilitate patient counselling and individualised treatment.

The impact of baseline serum C-reactive protein and C-reactive protein kinetics on the prognosis of metastatic nasopharyngeal carcinoma patients treated with palliative chemotherapy.

BACKGROUND: The aim of this study was to determine whether baseline C-reactive protein (CRP) levels and CRP kinetics predict the overall survival in metastatic nasopharyngeal carcinoma (mNPC) patients. METHODS: A total of 116 mNPC patients from January 2006 to July 2011 were retrospectively reviewed. Serum CRP level was measured at baseline and thereafter at the start of each palliative chemotherapy cycle for all patients. RESULTS: Patients with higher values of baseline CRP (≥ 3.4 mg/L) had significantly worse survival than those with lower baseline CRP values (< 3.4 mg/L). Patients were divided into four groups according to baseline CRP and CRP kinetics: (1) patients whose CRP < 3.4 mg/L and never elevated during treatment; (2) patients whose CRP < 3.4 mg/L and elevated at least one time during treatment; (3) patients whose CRP ≥ 3.4 mg/L and normalized at least one time during treatment; and (4) patients whose CRP ≥ 3.4 mg/L and never normalized during treatment. The patients were further assigned to non-elevated, elevated, normalized, and non-normalized CRP groups. Overall survival rates were significantly different among the four groups, with three-year survival rates of 68%, 41%, 33%, and 0.03% for non-elevated, elevated, normalized, and non-normalized CRP groups respectively. When compared with the non-elevated group, hazard ratios of death were 1.69, 2.57, and 10.34 in the normalized, elevated, and non-normalized groups (P < 0.001). CONCLUSIONS: Baseline CRP and CRP kinetics may be useful to predict the prognosis of metastatic NPC patients treated with palliative chemotherapy and facilitate individualized treatment. A prospective study to validate this prognostic model is still needed however.