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Abstract Background Previous investigations of transcriptomic signatures of cancer patient survival and post-therapy relapse have focused on tumor tissue. In contrast, here we show that in colorectal cancer (CRC) transcriptomes derived from normal tissues adjacent to tumors (NATs) are better predictors of relapse. Results Using the transcriptomes of paired tumor and NAT specimens from 80 Korean CRC patients retrospectively determined to be in recurrence or nonrecurrence states, we found that, when comparing recurrent with nonrecurrent samples, NATs exhibit a greater number of differentially expressed genes (DEGs) than tumors. Training two prognostic elastic net-based machine learning models—NAT-based and tumor-based in our Samsung Medical Center (SMC) cohort, we found that NAT-based model performed better in predicting the survival when the model was applied to the tumor-derived transcriptomes of an independent cohort of 450 COAD patients in TCGA. Furthermore, compositions of tumor-infiltrating immune cells in NATs were found to have better prognostic capability than in tumors. We also confirmed through Cox regression analysis that in both SMC-CRC as well as in TCGA-COAD cohorts, a greater proportion of genes exhibited significant hazard ratio when NAT-derived transcriptome was used compared to when tumor-derived transcriptome was used. Conclusions Taken together, our results strongly suggest that NAT-derived transcriptomes and immune cell composition of CRC are better predictors of patient survival and tumor recurrence than the primary tumor.

Abstract Background The 2016 World Health Organization (WHO) classification introduced nodal lymphomas of T follicular helper (Tfh) cell origin, such as angioimmunoblastic T-cell lymphoma (AITL), follicular peripheral T-cell lymphoma (F-PTCL), and nodal peripheral T-cell lymphoma with T follicular helper phenotype (nodal PTCL with TFH phenotype). However, the accurate incidence rate and clinical characteristics of F-PTCL and nodal PTCL with TFH are unstudied. Methods Between February 2012 to June 2020, a total of 207 cases diagnosed with nodal lymphomas of T follicular helper (Tfh) cell origin and PTCL-NOS were reviewed for clinical and histopathologic data. PTCL-NOS was defined to not correlate to any of the specific entities of mature T cell lymphoma in the WHO 2016 classification. We attempted to classify PTCL-GATA3 and PTCL-TBX21 by IHC staining. Target gene analysis was performed on a few patients with sufficient blood and tissue samples additionally. Results Among 207 patients, 111 patients (53.6%) had AITL, 67 patients (32.4%) had PTCL-NOS, 19 patients (9.2%) had F-PTCL, and 10 patients (4.8%) had nodal PTCL with TFH phenotype. We re-defined and analyzed F-PTCL and nodal PTCL with TFH phenotype into other TFH lymphomas. AITL (N = 101/111, 91.0%) was found to have a higher frequency of stage III/IV cancers compared to other TFH lymphomas (N = 22/29, 75.0%) and PTCL-NOS (N = 53/67, 79.1%; p-value = 0.03). The OS of AITL and other TFH lymphomas was similarly superior to PTCL-NOS (p-value = 0.02). AITL and other TFH lymphomas showed the TBX21 subtype more commonly than the GATA3 subtype. Mutations related to the RAS family (RHOA) and those related to epigenetic regulators (IDH2, DNMT3A, and TET2) were shown mainly in AITL and other TFH lymphomas. Conclusions Other TFH lymphomas appear to be a rare disease entity around one-quarter in nodal lymphomas of T follicular helper (Tfh) cell origin. Their less aggressive clinical feature than we did not expect is utterly different from PTCL-NOS and AITL. On the other hand, other TFH lymphomas share some characteristics, such as the cell of origin, a more common TBX21 subtype, and genetic variation such as RAS family mutation and epigenetic regulators, with AITL.

Abstract Background Targeted deep sequencing is increasingly used to detect low-allelic fraction variants; it is therefore essential that errors that constitute baseline noise and impose a practical limit on detection are characterized. In the present study, we systematically evaluate the extent to which errors are incurred during specific steps of the capture-based targeted sequencing process. Results We removed most sequencing artifacts by filtering out low-quality bases and then analyze the remaining background noise. By recognizing that plasma DNA is naturally fragmented to be of a size comparable to that of mono-nucleosomal DNA, we were able to identify and characterize errors that are specifically associated with acoustic shearing. Two-thirds of C:G > A:T errors and one quarter of C:G > G:C errors were attributed to the oxidation of guanine during acoustic shearing, and this was further validated by comparative experiments conducted under different shearing conditions. The acoustic shearing step also causes A > G and A > T substitutions localized to the end bases of sheared DNA fragments, indicating a probable association of these errors with DNA breakage. Finally, the hybrid selection step contributes to one-third of the remaining C:G > A:T and one-fifth of the C > T errors. Conclusions The results of this study provide a comprehensive summary of various errors incurred during targeted deep sequencing, and their underlying causes. This information will be invaluable to drive technical improvements in this sequencing method, and may increase the future usage of targeted deep sequencing methods for low-allelic fraction variant detection.

Abstract Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells.

Abstract Background Our study aims to evaluate the genetic and phenotypic spectrum of Frontotemporal dementia (FTD) gene variant carriers in Chinese populations, investigate mutation frequencies, and assess the functional properties of TBK1 and OPTN variants. Methods Clinically diagnosed FTD patients underwent genetic analysis through exome sequencing, repeat-primed polymerase chain reaction, and Sanger sequencing. TBK1 and OPTN variants were biologically characterized in vitro using immunofluorescence, immunoprecipitation, and immunoblotting analysis. The frequencies of genes implicated in FTD in China were analyzed through a literature review and meta-analysis. Results Of the 261 Chinese FTD patients, 61 (23.4%) carried potential causative variants in FTD-related genes, including MAPT (n = 17), TBK1 (n = 7), OPTN (n = 6), GRN (n = 6), ANXA11 (n = 4), CHMP2B (n = 3), C9orf72 GGGGCC repeats (n = 2), CYLD (n = 2), PRNP (n = 2), SQSTM1 (n = 2), TARDBP (n = 2), VCP (n = 1), CCNF (n = 1), CHCHD10 (n = 1), SIGMAR1 (n = 1), CHCHD2 (n = 1), FUS (n = 1), TMEM106B (n = 1), and UBQLN2 (n = 1). 29 variants can be considered novel, including the MAPT p.D54N, p.E342K, p.R221P, p.T263I, TBK1 p.E696G, p.I37T, p.E232Q, p.S398F, p.T78A, p.Q150P, p.W259fs, OPTN p.R144G, p.F475V, GRN p.V473fs, p.C307fs, p.R101fs, CHMP2B p.K6N, p.R186Q, ANXA11 p.Q155*, CYLD p.T157I, SQSTM1 p.S403A, UBQLN2 p.P509H, CCNF p.S160N, CHCHD10 p.A8T, SIGMAR1 p.S117L, CHCHD2 p.P53fs, FUS p.S235G & p.S236G, and TMEM106B p.L144V variants. Patients with TBK1 and OPTN variants presented with heterogeneous clinical phenotypes. Functional analysis demonstrated that TBK1 I37T and E232Q mutants showed decreased autophosphorylation, and the OPTN phosphorylation was reduced by the TBK1 I37T mutant. The OPTN-TBK1 complex formation was enhanced by the TBK1 E696G mutant, while OPTN R144G and F475V mutants exhibited reduced recruitment to autophagosomes compared to the wild-type. The overall frequency of TBK1 and OPTN in Chinese FTD patients was 2.0% and 0.3%, respectively. Conclusions Our study demonstrates the extensive genetic and phenotypic heterogeneity of Chinese FTD patients. TBK1 mutations are the second most frequent cause of clinical FTD after MAPT in the Chinese.