Significant Difference In Mutation Rates Research Articles

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

Understanding the heterogeneity in liver hepatocellular carcinoma with a special focus on malignant cell through single-cell analysis

IntroductionHepatocellular carcinoma (HCC) is the most common form of liver cancer globally and remains a major cause of cancer-related deaths. HCC exhibits significant intra-tumoral and interpatient heterogeneity, impacting treatment efficacy and patient prognosis.MethodsWe acquired transcriptome data from the TCGA and ICGC databases, as well as liver cancer chip data from the GEO database, and processed the data for subsequent analysis. We also obtained single cell data from the GEO database and performed data analysis using the Seurat package. To further investigate epithelial cell subgroups and their copy number variations, we used the Seurat workflow for subgroup classification and the InferCNV software for CNV analysis, utilizing endothelial cells as a reference. Pseudo-time analysis and transcription factor analysis of epithelial cells were performed using the monocle2 and SCENIC software, respectively. To assess intercellular communication, we employed the CellChat package to identify potential ligand-receptor interactions. We also analyzed gene expression differences and conducted enrichment analysis using the limma and clusterProfiler packages. Additionally, we established tumor-related risk characteristics using Cox analysis and Lasso regression, and predicted immunotherapy response using various datasets.ResultsThe samples were classified into 23 clusters, with malignant epithelial cells being the majority. Trajectory analysis revealed the differentiation states of the malignant epithelial cells, with cluster 1 being in the terminal state. Functional analysis revealed higher aggressiveness and epithelial-mesenchymal transition (EMT) scores in cluster 1, indicating a higher propensity for metastasis. RBP4+ tumor cells were highly enriched with hypoxia process and intensive cell-to-cell communication. A prognostic model was established, and immune infiltration analysis showed increased infiltration in the high-risk group. TP53 demonstrated significant differences in mutation rate between the two risk groups. Validation analysis confirmed the up-regulation of model genes, including AKR1B10, ARL6IP4, ATP6V0B, and BSG in tumor tissues.ConclusionA prognostic model was established based on HCC malignant cell associated gene signature, displaying decent prognosis guiding effectiveness in the multiple cohorts. The study provided comprehensive insights into the heterogeneity and potential therapeutic targets of LIHC.

Correlation between common driver gene variations and clinicopathological typing in lung adenocarcinoma

Objective: To correlate the common driver gene variations in primary lung adenocarcinoma with their clinical characteristics and histopathological subtypes. Methods: There were 4 995 cases of primary lung adenocarcinoma diagnosed at Weifang People's Hospital of Shandong Province from January 2015 to December 2021 which were retrospectively analyzed. Among them 1 983 cases were evaluated for their histopathological subtype; 3 012 were analyzed for the correlation of their histopathological subtypes and corresponding driver gene variations, including invasive non-mucinous adenocarcinoma (INMA) and invasive mucinous adenocarcinoma (IMA), and morphologically, poorly-differentiated, moderately-differentiated and well-differentiated adenocarcinomas. Next-generation sequencing was used to detect variations in EGFR, KRAS, ALK, RET, ROS1, MET, HER2, or BRAF driver genes. Results: There were 2 384 males and 2 611 females. EGFR and ALK variations were more commonly found in female patients aged 60 years or older, with EGFR mutation rate in clinical stage Ⅰ (25.80%) significantly higher than in other stages (P<0.05). KRAS mutations were more commonly detected in male smokers aged 60 years or older, HER2 mutations were more commonly in patients younger than 60 years, and RET mutations were more commonly in non-smokers (all P<0.05). No correlation was found between ROS1, MET, and BRAF gene variations and their clinical characteristics (P>0.05). For the histopathological subtypes, among the 1 899 cases of acinar adenocarcinoma, EGFR mutation rate was the highest (67.30%) compared to the other genes. Exon 21 L858R and exon 19 del were the main mutation sites in IMA and INMA, with a higher mutation rate at exon 20 T790M (11.63%) in micropapillary adenocarcinoma. In IMA, KRAS had the highest overall mutation rate (43.80%), with statistically significant difference in mutation rates of exon 2 G12D and exon 2 G12V in acinar adenocarcinoma, solid, and IMA (P<0.05). KRAS mutation at various sites were higher in poorly differentiated groups compared to moderately- and well-differentiated groups (P<0.05). HER2 mutations were more commonly observed in acinar adenocarcinoma, papillary, and micropapillary adenocarcinoma of INMA. BRAF mutation was higher in micropapillary adenocarcinoma compared with other types (P<0.05). Conclusions: Variations in EGFR, ALK, KRAS, HER2, and RET in primary lung adenocarcinoma are associated with patients' age, smoking history, and clinical stage, and driver gene mutations vary among different histopathological subtypes. EGFR mutations are predominant in INMA, while KRAS mutations are predominant in IMA.

Mutation Characteristics of inhA and katG Genes in Isoniazid-Resistant Mycobacterium Tuberculosis Patients in Xinjiang

Objective: To analyze the mutation characteristics of inhA and katG genes in isoniazid-resistant Mycobacterium tuberculosis in Xinjiang. Methods: The katG and inhA in 148 strains of isoniazid-resistant Mycobacterium tuberculosis were amplified through fluorescence quantitative PCR, and the amplified products were sequenced and compared. Results:The inhA gene mutation rate of 148 strains of isoniazid-resistant mycobacterium tuberculosis was 13.51% (20/148), among which the inhA gene mutation rate among patients of Han, Uygur, and Kazakh ethnicity were 15.87%, 13.21%, and 17.65%, respectively. There was no significant difference in the inhA mutation rate among nationalities (c2 = 2.897, P &gt; 0.05). The mutation rate of the katG gene was 84.46% (125/148), among which the mutation rates of patients of Han, Uyghur, and Kazak ethnicities were 82.54%, 84.91%, and 76.47%, respectively. The Hui and other ethnic groups were all affected by the katG gene mutation. There was no significant difference in the mutation rate of the katG gene among different ethnicities (c2 = 3.772, P &gt; 0.05). The mutation rates of the inhA gene in southern Xinjiang, northern Xinjiang, and other provinces were 18.60%, 9.28%, and 37.50%, respectively. The mutation rates of the inhA gene in different regions were statistically different (c2 = 6.381, P &lt; 0.05). There was no significant difference in the inhA mutation rate between patients from southern and northern Xinjiang (c2 = 2.214, P &gt; 0.05) and between southern Xinjiang and other provinces (c2 =1.424, P &gt; 0.05). However, the mutation rate of the inhA gene in patients from other provinces was higher than that in northern Xinjiang (c2 = 5.539, P &lt; 0.05). The mutation rates of the katG gene in southern Xinjiang, northern Xinjiang, and other provinces were 81.40%, 87.63%, and 62.50%, respectively. There was no significant difference in the mutation rates of the katG gene among different regions (c2 = 3.989, P &gt; 0.05). Conclusion: katG gene mutation was predominant in isoniazid-resistant tuberculosis patients in Xinjiang Uygur Autonomous Region, and inhA and katG gene mutation were no different among different ethnic groups.

The insights of mutations of gynecological oncology by liquid biopsy for Chinese patients.

e17501 Background: Liquid biopsy drew dramatic attention in the field of cancer research because of its attribution of nontraumatic, easy to obtain, providing overall state of tumor, allowing for real-time monitoring and so on. However, the transformation from research to application of liquid biopsy technique in clinical gynecological oncology management require more evidence and further investigation. Methods: Blood samples of 104 patients were collected, of whom 33 were diagnosed as cervical cancer, 20 were diagnosed as endometrial cancer, and 51 were diagnosed as ovarian cancer. cfDNA were extracted from blood and examined by next-generation sequencing technique with Haplox HapOnco panel. cfDNA mutations were analyzed and compared with genomic mutations in tumor tissues in publications. Results: In cfDNA, there were 120 genes mutated in cervical cancer patients, 150 genes mutated in endometrial cancer patients, and 58 genes mutated in ovarian cancer patients. Detecting by cfDNA, the average number of mutations was 6 (4.1 to 7.9, 95% CI) for each cervical cancer patient, 8.1 (2.1 to 14.1, 95% CI) for each endometrial cancer patient, and 5.1 (3.7 to 6.5, 95% CI) for each ovarian cancer patient. As the mutations in cfDNA compared with those in tumor tissues, the results revealed that there was no significant difference in mutation rate for 94.2% genes in blood and tumor tissues for cervical cancer patients, 93.3% for endometrial cancer patients, and 98.3% for ovarian cancer patients. Besides, there were no significant differences in microsatellite instability rates in blood and tumor tissues for cervical cancer patients and endometrial cancer patients. Conclusions: Most mutated genes in tumor tissues were detected in blood for gynecological oncology patients. The mutation rate for most genes and microsatellite instability rate had no significant difference in blood and tumor tissues, which suggested that liquid biopsy using cfDNA is helpful in gynecological oncology management.

The mutational landscape of Chinese breast cancer by liquid biopsy.

e13117 Background: Breast cancer is the most common malignancy worldwide. The novel technique of liquid biopsy provides further prospect of breast cancer management. However, the clinical application of liquid biopsy for breast cancer remains clearly defined. Methods: Blood samples of 145 Chinese patients diagnosed as breast cancer were collected. cfDNA were analyzed by next-generation sequencing technique using Haplox HapOnco panel. The mutations of cfDNA were analyzed and compared with genomic mutations detected within tumor tissues of breast cancer patients. Results: In total, 327 genetic mutations were detected in 145 patients using cfDNA. The top 5 high frequent alterations were TP53(50.3%), PIK3CA (42.8%), SYNE1 (16.6%), ESR1 (10.3%), CSMD3 (9.0%). Within TP53 mutations, 13.7% were frame shift insertions or deletions, 5.5% were in-frame insertions or deletions, 9.6% were splice site mutations. Within PIK3CA mutations, 1.6% were in-frame insertions or deletions, 1.6% were copy number variations. The average number of mutations for each patient was 7.2 (5.5 to 9.0, 95% CI). The mutated genes in cfDNA were compared with those in tumor tissues in publications. 208 mutated genes in tumor tissues were also detected in cfDNA. There was no significant difference in mutation rate for 83.6% genes in blood and tumor tissues. Conclusions: Most mutated genes in tumor tissues of breast cancer patients could be detected in cfDNA. The mutation rate for most genes had no significant difference in blood and tumor tissues, which indicated that liquid biopsy using cfDNA is a promising technique to improve breast cancer management.

Advantages of analysing both pairwise SNV-distance and differing SNVs between Mycobacterium tuberculosis isolates for recurrent tuberculosis cause determination.

Endogenous reactivation and exogenous reinfection are two possible causes of recurrent tuberculosis (TB). However, in some cases, precise cause determination can be challenging. In this study, we used whole genome sequencing to determine pairwise SNV distances and detect differing SNVs in initial and subsequent isolates for recurrent TB cases when the first and second episodes were caused by Mycobacterium tuberculosis (Mtb) strains with an identical spoligotype pattern. In total, 104 Mtb isolates from 36 recurrent TB and 16 single TB episode patients were included in the study. Most isolate pairs belonged to the SIT1 (n=21), SIT42 (n=9), SIT53 (n=9), and SIT254 (n=7) spoligotypes, and in 27 cases, resistance to at least one anti-TB drug was found in either isolate. Drug susceptibility was more common in the recurrent TB patient cohort, and longitudinal single TB episode isolates were more prone to be drug-resistant (p=0.03), while the association between patient cohort and spoligotype was not statistically significant (p=0.07). The pairwise SNV-distance between the longitudinal single TB episode isolates was small (0-7 SNVs). Among the recurrent TB isolates, based on the high SNV-distance (38-273 SNVs), six reinfection cases (16.7%) were identified. This distance was small (<10 SNVs) in the remaining 30 isolate pairs. Further analysis of differing SNVs revealed that 22 (61.1%) cases could be classified as possible reactivation. Notably, despite the small distance of 2-7 SNVs, initial isolates of eight patients (22.2%) had several SNVs that were not found in the second isolates; therefore, these cases were classified as reinfection with a closely related Mtb strain. No statistically significant difference in the time interval between specimen collection in the reactivation and reinfection Mtb sample groups (p=0.13) or an association between recurrence cause and drug resistance status (p=0.62) or spoligotype (p=0.79) could be detected. The mycobacterial median mutation rate of longitudinal single TB episodes and possible reactivation isolate pairs (n=37) was 0.12 SNVs/genome/year (IQR 0-0.39), and in 18 cases (48.6%), it was equal to zero. No statistically significant differences in mutation rate were found between recurrent TB and longitudinal single TB episode isolates (p=0.087), drug-susceptible and resistant isolates (p=0.37) or isolates of Beijing and other genotype families (p=0.33). Furthermore, four cases of fluoroquinolone resistance development through the acquired SNVs in the gyrA gene were identified. To conclude, this study highlighted the complexity of recurrent episode cause determination and showed the usefulness of differing SNV identification in both Mtb isolates in such cases. Expected drug susceptibility was the only discriminative factor for recurrent TB episode-causing mycobacterial strains, while no differences between reactivation and reinfection sample groups could be identified.

Abstract 53: The copy number landscape of early stage ovarian high grade serous carcinoma

Abstract Background: High grade serous carcinoma (HGSC) is the most common and poorest prognosis subtype of ovarian cancer. The large majority of patients present with advanced (stage IIIC or IV) disease with a median survival of only 3-4 years. By contrast, approximately 10% patients are diagnosed with early stage disease, confined to the ovary/fallopian tube, of whom 90% are cured with surgery and platinum-based chemotherapy. Beyond TP53 mutation, classic activating oncogene mutations are rare in HGSC. Rather, HGSC is marked by extreme copy number (CN) abnormalities, and this complexity has prevented detailed understanding of the mutational processes underpinning outcomes in HGSC. We have used shallow whole genome sequencing (sWGS) to develop novel CN signatures that are able to deconvolute the complexity of HGSC genomes. However, nearly all genomics studies in HGSC have examined advanced stage disease, and little is known about the genomics of early stage HGSC - specifically, it is unclear whether these tumors are identified purely by chance or whether they represent a specific subset that does not metastasize. Hypotheses: We hypothesized that early stage HGSC has a distinct copy number landscape compared to stage IIIC/IV disease. Methods and results: We have identified 43 cases of FIGO stage I-IIA HGSC from the pathology archives of three large London Gynaecological Cancer Centres and 52 late-stage (stage IIIC-IV) cases from BriTROC-1 cohort. Median age at diagnosis was 61.3 years vs 62.3 years respectively. There were no significant differences in mutation rates of TP53 and BRCA1/2, and TP53 mutations were near-universal in both cohorts. We also did not find cohort-specific focal SCNA that could explain biological behavior. However, ploidy was significant higher in late-stage (median 3.0) than early-stage (median 1.9) samples. CN signature exposures were significantly different between early and late stage cohorts. Relative exposure of signature 3 was greater in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic. Summary:This project identifed that early stage and late stage HGSC have highly similar patterns of mutation and focal SCNA. However, genome-wide analysis indicates that the abnormalities seen in advanced disease are not present in early stage disease, which may represent a discrete subset with reduced metastatic potential. By identifying and characterizing the copy number signature changes, these data suggest that diagnosis at early-stage might reflect biological differences and not fortuitous chance. These data improve understanding of HGSC biology and may reveal potential new treatment strategies. Citation Format: Zhao Cheng, Hasan B. Mirza, Darren P. Ennis, Philip Smith, Lena Morrill Gavarró, Chishimba Sokota, Gaia Giannone, Teodora Goranova, Thomas Bradley, Anna Piskorz, Michelle Lockley, Baljeet Kaur, Naveena Singh, Laura A. Tookman, Jonathan Krell, Jackie McDermott, Geoff Macintyre, Florian Markowetz, James D. Brenton, Iain A. McNeish. The copy number landscape of early stage ovarian high grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 53.

Clonal Architecture of TET2 Mutations Influences the Clinical Phenotype of Patients with Myeloid Neoplasms

Somatic TET2 mutations (TET2MT) are frequently found in myeloid neoplasia (MN) with a frequency that increases with age, consistent with the presence of subclinical TET2MT clones in asymptomatic older controls. Despite the prominent pathophysiologic role they play based on their prevalence; phenotype-genotype and prognostic associations have not been reliably established. We hypothesized that the diversity of TET2MT with regard to their configuration, genetic topography, and sub-clonal context, result in differences in biological, morphological, and clinical features.We studied blood and bone marrow samples from 4930 patients with MN using deep sequencing for common myeloid somatic mutations, including TET2 . This analysis identified 1205 TET2MT patients with 1781 distinct TET2MT, as many had more than one TET2MT. The most common TET2MT were frame shift (47%) and nonsense (34%) mutations, both gene truncating events. Truncations were subdivided into those occurring upstream enough to effectively delete the whole gene (46%) and those that only led to deletion of the catalytic domain. TET2MT were found in 17% of patients with MDS, 46% with MDS/myeloproliferative neoplasm (MDS/MPN), 19% with MPN, 21% with pAML and 24% with sAML. Across all MN's, single mutations were more common than biallelic or homozygous, except in MDS/MPN, where there were similar proportions of single and multiple mutant cases (22% single vs . 24% biallelic/homozygous TET2).TET2MT co-occurred most commonly with another TET2MT (43%), ASXL1 (21%), SRSF2 (18%), and NPM1 (13%). A similar distribution was seen in MDS, but with SF3B1 replacing NPM1 ; and in MDS/MPN, but with SF3B1 replaced by RUNX1 and CBL . In sAML the distribution of sub clonal lesions was similar to pAML, though with more mutations in NPM1 and FLT3ITD/TKD in pAML. Genes with the most significant differences in mutation rates between TETMTvs . TET2WT MDS/MPN patients were increased SRSF2 (34% vs . 15%, p<.001) and KRAS (8% vs . 3%, p<.001).The rank of TET2MT within the clonal hierarchy is heterogeneous. TET2MT were found to be ancestral hits in 40% of mutant cases. A second TET2MT was the most common secondary hit following an ancestral TET2 hit. Other common 2nd hits included mutations in SRSF2, ASXL1, DNMT3A, and SF3B1 . TET2MTirrespective of ancestral or secondary configurations had no impact on survival outcome. However, when comparing TET2MT clonal size, TET2 allelic burden negatively affected survival (p=.016) for all patients with MNs.Having ascertained that the majority of TET2 lesions are ancestral hits, we set to determine their impact on progression, extent of dysplasia, and proliferation (monocytosis, increase megakaryocytes fibrosis). We used logistic regression to assess odds ratios (OR) of a gene being associated with MDS vs. MDS/MPN. We then determined univariate Cox regression hazard ratios for each gene and used these to construct 2-dimensional regression plots (Figure 1). This plot shows that TET2MT as a sole ancestral hit implies a greater likelihood of dysplasia and less of progression to high risk (HR) disease as compared to low risk (LR). Subsequent secondary hits change the features of the disease as shown in Figure 1. A second (biallelic) hit in TET2 increased the odds for the disease being myeloproliferative (OR=1.17), a secondary U2AF1 hit increased the odds of progression (OR=2.64), and a sub clonal SRSF2 mutation increased both (OR=6.61, 3.89). RAS family mutations had a moderate effect on progression (OR=2.66, 1.71), but induced proliferative features (OR=8.77, 7.80), whereas SF3B1 mutations associated with dysplasia and LR (OR=0.26, 0.32). In comparison, all combined TET2 hits (irrespective of the secondary context) were plotted as a circle (Figure 1).In sum, our analyses demonstrate the genotypic/phenotypic impact of primary TET2 hits and how each subsequent mutation can affect the clinical phenotype of the disease. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Systematic Analysis of the Prognostic Impact of Somatic Mutations in Diffuse Large B-Cell Lymphoma (DLBCL) with Evaluation of Cell-of-Origin Dependence: Results from the Phase 3 GOYA Trial in Previously Untreated DLBCL

Introduction: Diffuse large B-cell lymphoma (DLBCL) represents a biologically and clinically heterogeneous diagnostic category with well-defined cell-of-origin (COO) subtypes, each arising from different stages of normal B-cell development. While the prognostic impact of the distinct COO subtypes has been confirmed in several studies, including the GOYA trial (NCT01287741; Vitolo et al. J Clin Oncol 2017), the impact of the key genomic alterations and their dependence on COO are less clear. Using data from the GOYA study, the largest prospective randomized trial in previously untreated DLBCL, we characterized the mutational profile of DLBCL by targeted next-generation sequencing (NGS), and evaluated the prognostic impact of somatic mutations and its relation to COO.Methods: Diagnostic formalin fixed paraffin-embedded tissue biopsies collected from patients (pts) with previously untreated DLBCL were tested at central laboratories. COO was assessed using the NanoString Research Use Only Lymphoma Subtyping (LST) gene expression assay (NanoString Technologies, Inc., Seattle, WA, USA). COO was evaluable in 933 pts (66% of the GOYA intent-to-treat [ITT] population of 1418 pts). Targeted DNA NGS was performed using the Foundation One Heme™ platform (all coding exons of 465 genes, select rearrangements in 31 genes) in 499 pts (35% of the ITT population). NGS and COO were available for 482 pts (34% of the ITT population). Only genetic alterations/mutations (single nucleotide variants [SNVs], copy number abnormalities [CNAs], chromosomal translocations) with known somatic and functional status were included in the statistical analysis, which was conducted as previously described (Frampton et al. Nat Biotechnol 2013). Multivariate Cox-regression was used to evaluate the prognostic effect of gene alterations with multiple testing adjustment using the Benjamini-Hochberg false-discovery rate (FDR) procedure (“significance” <5% FDR).Results: Baseline patient and disease characteristics were similar between pts with COO and/or NGS available, and the overall ITT population, except for geographic region and race. According to COO, 272/482 (56.4%) pts had germinal center B-cell-like (GCB), 78/482 (16.2%) unclassified, and 132/482 (27.4%) activated B-cell-like (ABC) DLBCL, which was similar to the overall COO distribution in GOYA. Of the 465 genes analyzed, we identified 59 genes with mutations (13%) occurring in at least 10/499 pts (≥2% prevalence), with an additional 334/465 genes with mutations occurring in at least 1 pt. The median number of gene alterations per pt was 6 (range 0-17). Overall, 3% of pts had no identified mutation and 93% of pts had multiple alterations (≥2 gene mutations). SNVs were the most common mutation type, while CNAs were specific to a few genes, including CDKN2A/B and REL (Figure 1A). Genes with significant differences in mutation rate (<5% FDR) between the GCB and ABC subtypes are shown in Figure 1B.On multivariate analysis, only alterations in BCL2 were associated with shorter progression-free survival (PFS) independent of COO, International Prognostic Index (IPI), treatment arm and number of planned chemotherapy cycles (Table 1). This effect was observed for both BCL2 translocations and BCL2 SNVs (Table 1; Figure 2), although the vast majority of pts with BCL2 SNVs also harbored BCL2 translocations (n=29/39; 74%), possibly explaining the negative prognostic impact of BCL2 SNVs. BCL2 alterations were detected in 102/499 pts, with 92/102 having BCL2 translocations, 90% (83/92) of which had GCB subtype DLBCL. Of the 39 pts with BCL2 SNVs, 80% (31/39) had GCB subtype and 15% (6/39) had ABC subtype DLBCL. BCL2 alterations were also significantly correlated with BCL2 gene and protein expression levels.Conclusions: Our data confirm the molecular heterogeneity of DLBCL, with potential treatment targets harbored by the distinct COO subtypes. Only alterations in BCL2 were associated with clinical outcome independently of of other factors (COO, IPI). [Display omitted] DisclosuresBolen:Genentech: Employment, Equity Ownership. Klanova:F. Hoffmann-La Roche Ltd: Employment, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party editorial support, under the direction of Magdalena Klanova, was provided by Lynda McEvoy of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd. Trněný:BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Sehn:Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Vitolo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Gilead: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria. Oestergaard:F. Hoffmann-La Roche Ltd: Employment.

Screening and clinical significance of lymph node metastasis-related genes within esophagogastric junction adenocarcinoma.

BackgroundDespite recent improvements in treatment technologies, such as surgical resection and chemoradiotherapy, the prognosis of patients with esophagogastric junction adenocarcinoma (EJA) remains poor due to early lymph node metastasis. Since few studies have investigated genes associated with lymph node metastasis in EJA, we aimed to screen lymph node metastasis‐associated genes and clarify their expression status and prognostic significance in EJA.MethodsThe differential frequency of mutations between carcinoma and para‐carcinoma tissues from 199 cases with EJA was detected using targeted next‐generation sequencing (tNGS). Following a stratified analysis to determine that gender has no effect on the frequency of gene mutations, lymph node metastasis‐related genes, including CDK6, MET, NOTCH1, and LRP1B, were screened, and CDK6 and LRP1B were selected for further study as they displayed significant differences in mutation rates. Differences in their expression status were verified using immunohistochemical (IHC) staining in 18 CDK6‐ and 17 LRP1B‐mutated samples and a randomly matched control group.ResultstNGS revealed that CDK6 and LRP1B mutation frequencies were significantly different between EJA cases with (N ≥ 1) or without (N = 0) lymph node metastasis. In particular, CDK6 mutation frequency was expected less, whereas that of LRP1B was remarkably higher in cases with stage N0 than in those with stage N ≥ 1. IHC staining confirmed significant differences in CDK6 and LRP1B expression status between the study and control cohorts. Chi‐square tests revealed that a high CDK6 expression status correlated significantly with smoking history (p = 0.044), T stage (p = 0.035), N stage (p = 0.000), and advanced TNM stage (p = 0.001) in EJA, whereas a high LRP1B expression status only correlated with BMI (p = 0.013) and N stage (p = 0.000). Furthermore, as confirmed by survival status investigation, a high LRP1B expression status predicted good prognosis, and a high CDK6 expression status was an independent predictor of poor prognosis in patients with EJA.ConclusionsTaken together, the findings of this study demonstrate that a high CDK6 and LRP1B expression status promotes and inhibits lymph node metastasis in patients with EJA, respectively, suggesting that both CDK6 and LRP1B are significantly potential predictors of lymph node metastasis and prognosis in EJA.

Abstract 4161: Integrated analysis of whole exome sequencing and hormone receptor expression data in endometrioid ovarian cancer

Abstract Endometrioid ovarian carcinomas (EnOCs) account for 10% of OC diagnoses, representing distinct biological and clinical entities compared to other OC subtypes. We recently reported two molecular subgrouping studies using &amp;gt;100 WT1-negative EnOCs: the first identified molecular subgroups of disease by unsupervised analysis of hormone receptor expression patterns, reporting favorable disease-specific survival (DSS) in the PR-high subgroups (PR+/ER+, PR+/ER- vs PR-/ER+, PR-/ER-). The latter study performed whole exome sequencing, identifying genomic events associated with differential DSS, including TP53 mutation (TP53m) and CTNNB1m. Here, we perform integrated analysis of these data to investigate the overlap between these molecular subgrouping layers. The PR-high subgroups demonstrated low TP53m rate (5% vs 26%, P=0.035) and frequent CTNNB1m (75% vs 26%, P&amp;lt;0.001), with the highest CTNNB1m rate in the PR+/ER+ subgroup (84%, 21/25). Accordingly, TP53m cases demonstrated significantly lower median histoscore (mHS) for PR (3 vs 203, P=0.028) and a trend for lower ER (mHS 66 vs 160, P=0.056), while cases with CTNNB1m demonstrated significantly higher PR and ER expression (mHS 258 vs 40, P&amp;lt;0.001 and 181 vs 75, P=0.002). There was no significant difference in tumor mutational burden, microsatellite instability or tumor heterogeneity as defined by Mutant Allele Tumour Heterogeneity (MATH) score between the hormone-receptor-based subgroups. These groups also demonstrated no significant difference in mutation rate of mismatch repair protein-encoding genes. Multivariable analysis identified only FIGO stage at diagnosis (P=0.002), optimal surgical debulking (P=0.035) and hormone receptor subgroup (P=0.004) as independently associated with DSS. TP53m and CTNNB1m were not independently associated with DSS (P=0.187 and P=0.166), though CTNNB1m demonstrated a trend for association in a corresponding model for relapse-free survival (RFS) (HR=0.26, P=0.064). Within PR-high cases, specific genomic events were not associated with significantly differential survival. Conversely, TP53m was associated with inferior outcome specifically in PR-low cases (P=0.010 for RFS; P=0.066 for DSS). Collectively, these data represent the first depiction of the overlay and interplay between recently identified EnOC subgroups defined by WES and hormone receptor expression patterns. They demonstrate that PR-high cases frequently harbor CTNNB1m, while TP53m is rare in this context. Moreover, they suggest that PR-high cases experience favorable outcome irrespective of genomic profile, while TP53m represents an important marker of prognosis in PR-low EnOC. Moreover, given the high expression of ER and PR in the CTNNB1m population, assessment of the potential efficacy of endocrine therapy in this population may now warrant investigation. Citation Format: Robert L. Hollis, John Thomson, Barbara Stanley, Alison M. Meynert, Michael Churchman, Tzyvia Rye, C. Simon Herrington, Charlie Gourley. Integrated analysis of whole exome sequencing and hormone receptor expression data in endometrioid ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4161.

Transcriptome analysis of testis reveals the effects of developmental exposure to bisphenol a or 17α-ethinylestradiol in medaka (Oryzias latipes)

Endocrine disrupting chemicals (EDCs) can induce abnormalities in organisms via alteration of molecular pathways and subsequent disruption of endocrine functions. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are ubiquitous EDCs in the environment. Many aquatic organisms, including fish, are often exposed to varying concentrations of BPA and EE2 throughout their lifespan. Both BPA and EE2 can activate estrogenic signaling pathways and cause adverse effects on reproduction via alteration of pathways associated with steroidogenesis. However, transcriptional pathways that are affected by chronic exposure to these two ubiquitous environmental estrogens during embryonic, larval, and juvenile stages are not clearly understood. In the present study, we examined transcriptional alterations in the testis of medaka fish (Oryzias latipes) chronically exposed to a low concentration of BPA or EE2. Medaka were exposed to BPA (10 μg/L) or EE2 (0.01 μg/L) from 8 h post-fertilization (as embryos) to adulthood 50 days post fertilization (dpf), and transcriptional alterations in the testis were examined by RNA sequencing (RNA-seq). Transcriptomic profiling revealed 651 differentially expressed genes (DEGs) between BPA-exposed and control testes, while 1475 DEGs were found between EE2-exposed and control testes. Gene ontology (GO) analysis showed a significant enrichment of “intracellular receptor signaling pathway”, “response to steroid hormone” and “hormone-mediated signaling pathway” in the BPA-induced DEGs, and of “cilium organization”, “microtubule-based process” and “organelle assembly” in the EE2-induced DEGs. Pathway analysis showed significant enrichment of “integrin signaling pathway” in both treatment groups, and of “cadherin signaling pathway”, “Alzheimer disease-presenilin pathway” in EE2-induced DEGs. Single nucleotide polymorphism (SNP) and insertion-deletion (Indel) analysis found no significant differences in mutation rates with either BPA or EE2 treatments. Taken together, global gene expression differences in testes of medaka during early stages of gametogenesis were responsive to chronic BPA and EE2 exposure.

Antimicrobial Resistance And Molecular Characteristics Among Neisseria gonorrhoeae Clinical Isolates In A Chinese Tertiary Hospital.

PurposeThe resistance of N. gonorrhoeae to antimicrobial agents has been increasing year by year due to the overuse of antibiotics. The primary aims of the present study were to investigate the molecular characteristics of the clinical isolates of Neisseria gonorrhoeae and the resistance to azithromycin in a Chinese tertiary hospital.MethodsFrom January 2014 to May 2017, a total of 55 clinical isolates of N. gonorrhoeae were collected. Genes associated with azithromycin resistance (AZM-R), including mutations in 23S rRNA alleles, the mtrR promoter and coding regions, and rplD and rplV were evaluated by PCR and DNA sequencing. All clinical isolates were subjected to N. gonorrhoeae multiantigen sequence typing (NG-MAST), while the AZM-R isolates were further characterized by multilocus sequence typing (MLST).ResultsThe AZM-R rate in this study was 23.64% (13/55), and a single (A)-nucleotide deletion mutation in the mtrR promoter region, a G45D mutation in the mtrR coding region, a point mutation in rplD, and an A2047G mutation in 23S rRNA alleles were detected in 13, 4, 3 and 4 isolates, respectively; no mutations were found in rplV. There was no significant difference in the mtrR coding region mutation rate between the azithromycin-sensitive and AZM-R groups (P > 0.05); however, there was a significant difference in the mutation rate of the mtrR promoter region (P < 0.05). Among the 55 isolates studied, 43 distinct NG-MAST were determined, while the AZM-R isolates were allocated into 10 distinct MLST/NG-MAST combinations. All three isolates with high-level AZM-R belonged to the sequence types (STs) NG-MAST ST1866 and MLST ST10899.ConclusionN. gonorrhoeae clinical isolates from Wenzhou, eastern China, showed considerable genetic diversity. Measures should be implemented to monitor the spread of the NG-MAST ST1866 and MLST ST10899 N. gonorrhoeae clones, which exhibit high-level AZM-R in eastern China.

Mutation analysis of short tandem repeats in a population sample from Upper Silesia (southern Poland).

In paternity testing, DNA polymorphism analysis not only settles explicitly disputed paternity issue but also provides information on mutation frequencies in STR loci. In this study, insertion or deletion of one repetitive unit was observed in 38 of 32,391 meiotic transfers analysed in 953 paternity testing cases. Parentage samples from Upper Silesia (southern Poland) were examined in 2008-2014 with the use of three commercially available amplification kits: AmpFlSTR Identifiler (Applied Biosystems), PowerPlex 16 HS (Promega) and PowerPlex ESX 17 (Promega). The rate of paternal mutations was 4.6 times higher than that of maternal ones. The highest mutation rate was noted at VWA locus. Interpopulation comparisons showed statistically significant differences in mutation rates of several STRs between Upper Silesia and populations from Brazil and China. There were no differences in occurrence of mutations between a population from Upper Silesia and another southern Polish population from a region of Lesser Poland. Our results suggest that knowledge of STR mutation rates in different populations may be important for calculations of probability of relationship in disputed paternity testing and that such calculations should be based on population-specific mutation rates, at least for some STR markers used commonly in forensic genetics.

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.

What about the guys? An assessment of gender differences in hereditary colorectal cancer testing.

537 Background: Approximately 5-10% of colorectal cancer (CRC) is due to hereditary causes. Identification of an inherited cause may impact surgical and treatment decisions for CRC patients and may identify increased risks for other cancers that warrant increased screening and/or risk reduction measures. Men have testing for hereditary breast and ovarian cancer less often than women, even though these genes may also cause increased risk for cancer in men and men are as likely as women to carry mutations in these genes and pass them onto their children. We aimed to explore whether similar gender differences exist related to testing for hereditary CRC. Methods: We retrospectively reviewed clinical data and test results from consecutive CRC cases, who had a multi-gene panel with 13-49 genes at our laboratory, between March 2012 and June 2016. Statistical comparisons between males and females were conducted using Fisher’s exact test. Results: Of CRC cases (n = 7142), 61.1% were female and 12.8% were positive for mutation or likely pathogenic variant. Average age of CRC onset for men was 47.2, and for women was 49.5. Women with CRC before age 20 had the highest mutation rate (31.8%), but men were more likely to test positive than women overall (14.1% vs 12.0%, p = 1.1e-2). Mutations were most frequent in the same three genes for both men and women, but in different orders: MLH1, CHEK2, and MSH2 for males and CHEK2, MSH2, and MLH1 for females. The only genes with significant differences in mutation rates between men and women were MLH1 (3.2% vs 1.5%, p = 2.0e-6) and MSH6(1.7% vs 0.8%, p = 3.0e-3). Conclusions: Hereditary CRC is expected to affect men and women equally. In our cohort, however, the majority of individuals tested were women, while men were more likely to test positive. Possible explanations include smaller sample size and earlier age of onset in men, perceptions of referring clinicians, and different levels of interest in genetic counseling and testing between male and female patients. These data highlight an important opportunity for educating and identifying more men with hereditary CRC who may benefit from genetic information. Further studies are needed to confirm these results and explore reasons for the differences.

880P - Molecular characterization and comparison of epithelial ovarian carcinoma (EOC) and primary peritoneal carcinoma (PCC)

EOC and PPC are two cancers currently treated in a similar manner. Molecular tumor profiling, with its emphasis on individualized therapy, is altering prior treatment paradigms by focusing on molecular aberrations rather than organ primary to guide therapy. Studies at our institution on EOC and PPC suggest these are two distinct cancer types, with PPC being more aggressive. The aim of this study is to identify further differences in the molecular profiles of EOC and primary peritoneal carcinoma in order to identify treatment/resistant mechanisms and clinical trial opportunities. In total, 5,685 EOC and 521 primary peritoneal carcinoma specimens were evaluated (Caris Life Sciences) by immunohistochemistry (IHC), in situ hybridization (ISH), fusion gene analysis, and next-generation sequencing (NGS). Initial diagnosis was made by the submitting institution and confirmation of diagnosis was made by a pathologist at the centralized laboratory. Significant differences were found between IHC and NGS. Protein expression in androgen receptor (28.8% v. 36.5%, p = 0.0011), EGFR (48.9% v. 59.0%, p = 0.0081), ER (46.0% v. 55.6%, p = 0.0001), PD-L1 (9.5% v. 5.7%, p = p = 0.0300), PR (22.5% v. 14.6%, p = 0.0001), TLE3 (18.4% v. 13.2% p = 0.0120), TOP2A (75.9% v. 66.8%, p = 0.0001), and TS (54.3% v. 44.1%, p = 0.0001) varied significantly between EOC and PPC, respectively. Significant differences in mutation rates were found in CTNNB1 (3.3% v. 0.7%, p = 0.0033), KRAS (9.4% v. 3.7%, p = 0.0001), PIK3CA (9.3% v. 4.8%, p = 0.0027), PTEN (3.9% v. 1.6%, p = 0.0222), and TP53 (63.3% v. 74.5%, p = 0.0001). No significant differences were found in amplification rates, as measured by ISH and CNV by NGS. Multiplatform profiling reveals various potential targets in ovarian and primary peritoneal carcinomas for investigational and drug therapy. Comparison of their genetic profiles reveals two distinct cancers. Dysregulation of the PIK3CA/AKT/mTOR pathway appears to be more common in EOC while loss of TP53 is a more common event in PPC based on this cohort. More studies are urgently needed to assess differences between EOC and PPC.

Comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice (Oryza sativa)

Rice seeds, after space flight and low dose heavy ion radiation treatment were cultured on ground. Leaves of the mature plants were obtained for examination of genomic/epigenomic mutations by using amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) method, respectively. The mutation sites were identified by fragment recovery and sequencing. The heritability of the mutations was detected in the next generation. Results showed that both space flight and low dose heavy ion radiation can induce significant alterations on rice genome and epigenome (P<0.05). For both genetic and epigenetic assays, while there was no significant difference in mutation rates and their ability to be inherited to the next generation, the site of mutations differed between the space flight and radiation treated groups. More than 50% of the mutation sites were shared by two radiation treated groups, radiated with different LET value and dose, while only about 20% of the mutation sites were shared by space flight group and radiation treated group. Moreover, in space flight group, we found that DNA methylation changes were more prone to occur on CNG sequence than CG sequence. Sequencing results proved that both space flight and heavy ion radiation induced mutations were widely spread on rice genome including coding region and repeated region. Our study described and compared the characters of space flight and low dose heavy ion radiation induced genomic/epigenomic mutations. Our data revealed the mechanisms of application of space environment for mutagenesis and crop breeding. Furthermore, this work implicated that the nature of mutations induced under space flight conditions may involve factors beyond ion radiation.

Comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice (Oryza sativa)

Rice seeds, after space flight and low dose heavy ion radiation treatment were cultured on ground. Leaves of the mature plants were obtained for examination of genomic/epigenomic mutations by using amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) method, respectively. The mutation sites were identified by fragment recovery and sequencing. The heritability of the mutations were detected in the next generation. Results showed that both space flight and low dose heavy ion radiation can induce significant alterations on rice genome and epigenome (P<0.05). For both genetic and epigenetic assay, while there was no significant difference in mutation rates and their ability to be inherited to the next generation, the site of mutations differed between the space flight and radiation treated groups. More than 50% of the mutation sites were shared by two radiation treated groups, radiated with different LET value and dose, while only about 20% of the mutation sites were shared by space flight group and radiation treated group. Moreover, in space flight group, we found that DNA methylation changes were more prone to occur on CNG sequence than CG sequence. Sequencing results proved that both space flight and heavy ion radiation induced mutations were wide spread on rice genome including coding region and repeated region. Our study described and compared the characters of space flight and low dose heavy ion radiation induced genomic/epigenomic mutations. Our data revealed the mechanisms of application of space environment for mutagenesis and crop breeding. Furthermore, this work implicated that the nature of mutations induced under space flight conditions may involve factors beyond ion radiation.