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Antitumor peptide vaccine is an alternative and attractive way to eradicate tumors by the aid of host immune system. The current peptide vaccine development is hampered by several hurdles. These include heterogeneity of tumor cells, applicable in vivo stability of peptides, and others. One of the major obstacles is the precise prediction of cancer antigen, which can be loaded efficiently onto antigen presenting cells. We present here a neoantigen prediction algorithm DeepNeo comprised of DeepNeo-MHC and DeepNeo-TCR, which calculates the antigenicity of neoantigen based on the MHC binding and TCR activation potential. We examined the efficacy of DeepNeo using ELISPOT and two allogenic mouse tumor models. By treating the combination of long peptides targeting MC38 colon or B16F10 melanoma cell lines, we have shown the DeepNeo effectively determines neoantigens for the development of antitumor peptide vaccine. Analysis of tumor-infiltrated lymphocytes (TIL) supported our results. Undergoing study using patient-derived xenograft model treated with autologous, trained PBMC with personalized peptide vaccine will be discussed further. Citation Format: Eun Ji Lee, Min Ji Park, Soo Jin Kim, Seung-Jae Noh, Inkyung Shin, Jeong Yeon Kim, Incheol Shin, Jung Kyoon Choi, Dae-Yeon Cho, Suhwan Chang. Efficacy analysis of the DeepNeo neoantigen prediction tool for antitumor vaccine development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB168.

Previous biomarkers such as PD-L1 expression, tumor mutation burden (TMB), and neoantigen load have limited accuracy in predicting responses to immune checkpoint inhibitor (ICI) therapy. In this work, we propose an improved predictor of ICI responses on the basis of our previous findings on the role of neoantigen functionality and genomic hypomethylation in tumor immunity. Specifically, we developed a metric of constitutive neoantigen load given the association of constitutive neoantigens derived from genes indispensable for cancer growth with favorable clinical responses to ICI (Clin. Transl. Med. 12:e714). A panel that captures somatic mutations on the 300 most essential genes identified by genome-wide screening in ~1,800 pan-cancer cell lines was used. Our DeepNeo algorithm (Nat. Commun. 11:951; Nat. Genet. 2023) was employed to identify T cell-reactive neoantigens from somatic mutations binding patient-matched HLA proteins. Also, considering the role of genomic hypomethylation in inducing resistance of tumors to ICI therapy (Nat. Commun. 10:4278), we developed an assay that estimates genomic hypomethylation by targeted sequencing of LINE-1 elements. Constitutive neoantigen load and LINE-1 methylation status were evaluated in our cohort consisting of 335 lung cancer patients treated with ICIs. As a result, constitutive neoantigen load (HR=0.671; p-value=3.13E-03) and LINE-1 methylation (HR=0.597; p-value=0.012) outperformed PD-L1 (HR=0.74; p-value=0.199), TMB (HR=0.917; p-value=0.511), and the combination of PD-L1 and TMB (HR=0.707; p-value=0.218). The combination of constitutive neoantigen load and LINE-1 methylation (HR=0.245; p-value=1.11E-05) showed even better predictive power. In conclusion, our approach can be used as new companion diagnostics for ICI-based immune-oncology. Citation Format: Hyoeun Bang, Jeong Yeon Kim, Cheolyong Joe, Kyeonghui Kim, Younghak Bang, Hyemin Kim, Honghi Cha, Eunjoo Oh, Naeun Lee, Inkyung Shin, Seung-Jae Noh, Dae-Yeon Cho, Se-Hoon Lee, Jung Kyoon Choi. Improved prediction of immunotherapy responses by constitutive neoantigen load and LINE-1 methylation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5475.

Objective: We investigated the interaction effect between the methylation of dopamine receptor D4 (DRD4) and phthalate exposure in ADHD on continuous performance test (CPT) variables. Method: Urine concentrations of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono-n-butyl phthalate (MBP) were tested. The methylation status was analyzed for CpG sites of DRD4. Multivariable linear regression models were applied to investigate the interaction effects of methylation and phthalate levels. Results: There was a significant interaction effect of the methylation of CpG26 and CpG28 with the MEHHP level on omission errors in ADHD patients, but not in controls. The post hoc analysis revealed a significant correlation between the MEHHP concentration and omission errors in the methylated group, but not in the unmethylated group. Conclusion: The interaction between the methylation status of CpG sites of DRD4, particularly CpG26 and CpG28, and phthalate metabolite levels affects the attention level in ADHD patients.

Purpose The aim of the current study is to assess the spectrum of genetic variation in the BRIP1 gene among Korean high-risk breast cancer patients who tested negative for the BRCA1/2 mutation. Materials and methods Overall, 235 Korean patientswith BRCA1/2 mutation-negative high-risk breast cancerwere screened for BRIP1 mutations. The entire BRIP1 gene was analyzed using fluorescent-conformation sensitive gel electrophoresis. In silico analysis of BRIP1 variants was performed using PolyPhen-2 and SIFT. Results A total of 20 sequence alterations including 12 exonic and eight intronic variantswere found. Among the 12 exonic variants, 10 were missense and two were silent mutations. No protein-truncating mutation was found among the tested patients. Among the 10 missense variants, four (p.L263F, p.L340F, p.L474P, and p.R848H) were predicted to be pathogenic by both PolyPhen-2 and SIFT, and these variants were found in five patients. Of the four missense variants, p.L263F, p.L474P, and p.R848H localize to regions between the helicase motifs, while p.L340F resides in an iron-sulfur domain of BRIP1. Conclusion No protein-truncating mutation in BRIP1 was found among the tested patients. The contribution of BRIP1 variants is thought to be minor in Korean non-BRCA1/2 high-risk breast cancer.

Environmental factors may interact with genetic factors via the epigenetic process, and this interaction can contribute to inter-individual variability in the treatment response. The purpose of this study was to investigate the interaction effects between dopamine receptor D4 (DRD4) methylation and prenatal maternal stress on the methylphenidate (MPH) response of youth with attention-deficit/hyperactivity disorder (ADHD).This study was an 8-week open-label trial of MPH that included 74 ADHD youth. We investigated the associations between MPH treatment response, which was defined as a score ≤2 on the Clinical Global Impressions-Improvement (CGI-I) scale, and the methylation of 28 cytosine-guanine dinucleotide (CpG) sites of DRD4. Additionally, the interaction effects between DRD4 methylation and prenatal maternal stress on changes in Continuous Performance Test (CPT) scores after MPH treatment were investigated.Although there were no significant sites that showed significant association with treatment response, there was a significant interaction effect of the methylation of CpG7 and prenatal maternal stress on changes in omission errors of the CPT following treatment (p = 0.0001).The present findings indicate that the interaction between methylation of CpG7 of DRD4 and prenatal maternal stress may be predictive of the treatment response to MPH in youth with ADHD.

Supplemental material, JAD_Supplementary_revision for Interaction of DRD4 Methylation and Phthalate Metabolites Affects Continuous Performance Test Performance in ADHD by Johanna Inhyang Kim, Jae-Won Kim, Inkyung Shin, and Bung-Nyun Kim in Journal of Attention Disorders

The purpose of the present study was to analyze genetic variations in the NBS1 gene and to evaluate the contribution of heterozygous NBS1 mutation to the risk of breast cancer among Korean patients with high-risk breast cancer negative for BRCA1/2 mutation. We screened 235 non-BRCA1/2 Korean patients with high-risk breast cancer for NBS1 mutations. The entire NBS1 gene was sequenced using fluorescent conformation-sensitive capillary electrophoresis. In silico analysis of the NBS1 variants was performed using PolyPhen-2 and SIFT. The frequency of variants predicted to be deleterious by in silico analysis was compared between breast cancer patients and controls. Twenty-eight sequence variants in the NBS1 gene were identified: 9 exonic variants, including 5 missense mutations (p.R169C, p.I171V, p.E185Q, p.E564K, and p.F603L) and 4 silent mutations, and 19 variants within introns. Among the five missense variants, p.I171V (c.511A > G) was the only variant predicted to be deleterious by in silico analysis. Heterozygosity for p.I171V was found in 4/235 patients with breast cancer and 3/281 individuals in the control group. The frequency of p.I171V was not significantly different between the patient and control groups (1.7 vs. 1.06 %, p = 0.7). Heterozygosity of p.I171V in the NBS1 gene was found in a small proportion of Korean patients with high-risk breast cancer. The contribution of the p.I171V variant to the development of breast cancer among Korean patients was not significant.

We screened large genomic rearrangements of the BRCA1 and BRCA2 genes in Korean, familial breast cancer patients. Multiplex ligation-dependent probe amplification assay was used to identify BRCA1 and BRCA2 genomic rearrangements in 226 Korean familial breast cancer patients with risk factors for BRCA1 and BRCA2 mutations, who previously tested negative for point mutations in the two genes. We identified only one large deletion (c.4186-1593_4676-1465del) in BRCA1. No large rearrangements were found in BRCA2. Our result indicates that large genomic rearrangement in the BRCA1 and BRCA2 genes does not seem like a major determinant of breast cancer susceptibility in the Korean population. A large-scale study needs to validate our result in Korea.

This study was performed to evaluate the frequency of mutations in CHEK2, PALB2, MRE11, and RAD50 among Korean patients at high risk for hereditary breast cancer.A total of 235 Korean patients with hereditary breast cancer who tested negative for BRCA1/2 mutation were enrolled to this study. Entire coding regions of CHEK2, PALB2, MRE11, and RAD50 were analyzed using massively parallel sequencing (MPS). Sequence variants detected by MPS were confirmed by Sanger sequencing.Six patients (2.5 %) were found to have pathogenic variants in CHEK2 (n = 1), PALB2 (n = 2), MRE11 (n = 1), and RAD50 (n = 2). Among the pathogenic variants, PALB2 c.2257CT was previously reported in other studies, while CHEK2 c.1245dupC, PALB2 c.1048CT, MRE11 c.1773_1774delAA, RAD50 c.1276CT, and RAD50 c.3811_3813delGAA were newly identified in this study. A total of 15 missense variants were found in the four genes among 26 patients; 7 patients had a variant in CHEK2, 11 in PALB2, 2 in MRE11, and 6 in RAD50. When in silico analyses were performed to the 15 missense variants, six variants (CHEK2 c.686AG, PALB2 c.1492GT, PALB2 c.3054GC, MRE11 c.140CT, RAD50 c.1456CT, and RAD50 c.3790CT) were predicted to be deleterious.Pathogenic variants in CHEK2, PALB2, MRE11, and RAD50 were detected in a small proportion of Korean patients with features of hereditary breast cancer.

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.

Purpose The aim of the current study is to assess the spectrum of genetic variation in the BRIP1 gene among Korean high-risk breast cancer patients who tested negative for the BRCA1/2 mutation. Materials and Methods Overall, 235 Korean patientswith BRCA1/2 mutation–negative high-risk breast cancerwere screened for BRIP1 mutations. The entire BRIP1 gene was analyzed using fluorescent-conformation sensitive gel electrophoresis. In silico analysis of BRIP1 variants was performed using PolyPhen-2 and SIFT. Results A total of 20 sequence alterations including 12 exonic and eight intronic variantswere found. Among the 12 exonic variants, 10 were missense and two were silent mutations. No protein-truncating mutation was found among the tested patients. Among the 10 missense variants, four (p.L263F, p.L340F, p.L474P, and p.R848H) were predicted to be pathogenic by both PolyPhen-2 and SIFT, and these variants were found in five patients. Of the four missense variants, p.L263F, p.L474P, and p.R848H localize to regions between the helicase motifs, while p.L340F resides in an iron-sulfur domain of BRIP1. Conclusion No protein-truncating mutation in BRIP1 was found among the tested patients. The contribution of BRIP1 variants is thought to be minor in Korean non-BRCA1/2 high-risk breast cancer.

This investigation is aimed at evaluating the epidemiologic characteristics of BRCA1/2 germline mutations in Korean patients with breast and ovarian cancer (BOC). We analyzed the entire mutational data of BRCA1/2 genes in BOC patients who were tested in Korea since the first Korean report of BRCA1 mutation in 1995 with the exception of the data covered in the Korean Hereditary Breast Cancer (KOHBRA) study, the project launched in 2007 for establishing BRCA1/2 carrier cohorts in Korea. In total, BRCA1/2 gene mutations of 3,922 Korean BOC patients were evaluated, including the unpublished data of 2,139 breast cancer patients examined by four Korean institutions and the data of 1,783 BOC patients covered in ten previous reports. Overall, 420 (150 distinct) pathogenic mutations were identified, 211 (73 distinct) in BRCA1 and 209 (77 distinct) in BRCA2. The majority (134 of 150) of the distinct mutations resulted in premature termination codon of the BRCA1/2 translation. BRCA1 c.4186-1593_4676-1465del was the only large genomic rearrangements mutation. Out of 150 distinct BRCA1/2 mutations, 84 (56 %) mutations were considered specific to Korean BOC. Eighty-five BRCA1/2 mutations were detected in at least two unrelated patients. These recurrent mutations account for 84.5 % (355 of 420) of mutations detected in the Korean population. In the pooled mutational data of BRCA1/2 genes, this study discovered the prevalence of BRCA1/2 mutations in the Korean BOC patients is similar to those found in other ethnic groups. Large genomic rearrangements in BRCA1/2 genes were infrequently detected among the Korean patients with BOC. There were several BRCA1/2 mutation candidates for founder mutations. To further establish a Korean cohort for BRCA1/2 mutations, the nationwide KOHBRA study is in progress.

Publisher Summary This chapter reviews the substantial parts of a genome that regulate the functions of DNA and RNA. Regulation of gene replication and expression critically depends on the sequence-specific binding proteins or ribonucleoprotein complexes in processes such as DNA synthesis, repair and transcription, and RNA editing, splicing, translation, and degradation. The chapter aims to provide a method for making all possible substitutions in every position of the phage SP6 promoter and measuring their effects on promoter activity in vivo and in vitro. The relative in vivo activity of promoter variants can be determined by measuring the interference to replication of promoter-bearing plasmids by transcription. This assay has an advantage over direct assays of RNA measurement or indirect assays for a reporter protein. The chapter discusses a graphic method called “activity logos,” used to display saturation mutagenesis data. Activity logos, based on energetics, provide a quantitative view of the energetic contribution of each residue to promoter function.

A full set of SP6 promoter variants with all possible single substitutions at positions −17 to +5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues −11, −9, −8, −7, and +1 (initiation site). All substitutions at −11, −9, −8, and −7 abolished formation of closed complexes, except for A−8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro . In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo . Some variants at −13, −4, and −2, among others, showed exceptionally higher activities in vivo than in vitro , supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A−3T variant showed much lower activity in vivo than in vitro , but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.