Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Expression and prognostic effect of DEPTOR in rectal carcinoma / 局解手术学杂志

Objective To clarify the expression of DEPTOR in rectal cancer,and to further explore the relationship between the expres-sion level of DEPTOR and histopathology and prognosis,in order to provide reference for the clinical diagnosis and treatment of rectal cancer. Methods The clinical data of 102 patients who underwent radical resection of rectal cancer in our hospital from January 2011 to January 2013 were analyzed retrospectively.The expression of DEPTOR in cancer tissues and adjacent tissues were evaluated by immunohistochemis-try and immunoblotting.The patients were divided into high expression group and low expression group by the median value of integrated opti-cal density(IOD);the relationship between the expression level of DEPTOR and clinical,histopathology and prognosis was analyzed. Results The results ofImmunohistochemistry and immunoblotting showed that the expression level of DEPTOR in cancer tissues was higher than that in adjacent tissues,the differences were significant(P<0.05). Univariate analysis showed that there was no significant differences in gender, age,and BMI(P>0.05),and there were significant differences in tumor diameter,T stage,N stage,and differentiation between the high-ex-pression and low-expression group (P<0.05).The independent influencing factors of DEPTOR expression was analyzed by the Logistic re-gression model,which showed that T stage and tumor diameter were independent influencing factors of high expression of DEPTOR.Compared with low expression group,the serum CEA level in patients with high expression group was higher,the differences was statistically significant ( P < 0.05 ). There was no significant difference in serum CA199 level between the expression group and the low expression group (P>0.05).Spearman correlation analysis showed that the expression level of DEPTOR was positively correlated with serum CEA level in rectal cancer patients (r=0.509,P<0.01).Compared with low expression group,the 5-year cumulative recurrence rate and the 5-year cumulative mortality rate in the high-expression group of DEPTOR were higher,the differences were statistically significant ( P <0.05). Conclusion DEPTOR is highly expressed and is associated with the degree of disease progression in rectal cancer,its elevation suggests a poor prognosis.

A study on the gene alterations in 9 STR loci and Amelogenin locus in cancerous tissues / 中国法医学杂志

Objective To study genetic alterations in 9 STR loci and the Amelogenin locus in various tumor tissues. Methods twenty cancer tissues taken from 20 different unrelated individuals and their blood specimens were examined with Chelex-100 extraction of DNA, Profiler Plus PCR amplification and 310 Genetic Analyzer. Results All of the 10 STR loci exist genetic alterations. The genetic alterations occurred in 6out of 20 cases. The rate of genetic alteration was 30%. Six genetic alterations were found in one tumor tissue. Conclusion The forensic community has to take be cautious not to use the tumor tissue for personnel identification.