Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma

Background: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wildtype cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. Methods: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C \u3e T and chr5:1,295,250 C \u3e T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations. Results: The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%- 79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases. Conclusions: The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers

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Not AvailableExtrusion cooking is a unique method for preparing pasta, which is generally produced from durum wheat semolina. However, preparation of pasta from sorghum is not practiced in India. Therefore, the present study was undertaken to develop and standardize pasta from sorghum cultivar, M35-1 and wheat semolina of 0.1 mm particle size. Sorghum and wheat semolina in different proportions (T1;S:W-50:50,T2;S:W-60:40,T3;S:W-70:30,T4; S:W-80:20, T5; S −100) were mixed with lukewarm water (40 °C) in the cold extruder for 30 min and passed through the extruder with a screw speed of 80 rpm and at a temperature of 55° to obtain pasta of diameter (0.6 mm) and length (1.4 mm). The extruded pasta was dried at 70 °C in a tray drier for 8 h, cooled and stored in polyethylene bags at room temperature. The pasta was subjected to physico-chemical analysis such as length, diameter, bulk density, water absorption, cooking time, cooking loss, moisture, water activity, alcoholic acidity, amylase, carbohydrates, fat, protein, fibre and ash using standard methods. Organoleptic characteristics such as color and appearance, texture, taste, flavor and overall acceptability, stickiness, bulkiness and firmness were evaluated at laboratory level by a panel of semi trained judges using 5 point hedonic rating scale. Among the various blends studied, the sorghum and wheat semolina with a combination of 50:50 (T1) and 60:40 (T2) and 70:30 (T3) were more acceptable than others. Well acceptable sorghum pasta can be developed from sorghum and wheat, thereby improving its nutritional composition.Not Availabl