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.

Monoclonal gammopathies and the related autoimmune manifestations in Taiwan.

A total of 50,000 patients were surveyed for the presence of monoclonal immunoglobulins during the past two decades. There were 411 cases of monoclonal gammopathies including 243 cases of plasma cell neoplasms and 168 cases of secondary plasma-cell dyscrasia. Among the 227 cases of multiple myeloma and Waldenström's macroglobulinemia, there were 49.3% IgG class, 22.9% IgA class, 9.7% IgM class and 13.2% light chain type. In addition, there were 1.3% of nonexcretory myeloma including an IgM type. A relatively high frequency (4.8%) of IgD M-proteins was detected but heavy chain disease was not encountered in the present series. Purified M-components from patients with possible autoimmune manifestations were subjected to immunofluorescence studies. Autoimmune activity of M-proteins was found in a patient of Waldenström's macroglobulinemia with peripheral neuropathy, and another patient of cryofibrinogenemia with recurrent purpura and gangrene. In conclusion, a high frequency of IgD myeloma is found in Chinese patients of this area. M-components may have autoimmune activity resulting in unusual clinical manifestations.