High Mutation Frequency Research Articles

Intratumoral heterogeneity as a predictive biomarker in immunotherapy for advanced bladder cancer.

e16619 Background: Previous studies have shown that intra-tumor heterogeneity (ITH) can lead to drug resistance to tumor therapy, which is associated with poor prognosis of solid tumors. However, studies on the correlation between ITH and immunotherapy efficacy in patients with advanced bladder cancer (BLCA) are still lacking. Methods: Bioconductor R package Maftools was used to analyze somatic variants of advanced BLCA from an independent cohort (Miao-249 study cohort, PMID: 30150660) with data from 27 patients who underwent whole-exome sequencing. The ITH score was measured using the mutant-allele tumor heterogeneity (MATH) algorithm to analyze the difference in the genetic profile between the ITH score high (ITH-H) and ITH score low (ITH-L) groups, its association with tumor mutational burden (TMB), and the effect of ITH on the prognosis of immunotherapy. Results: There were significant differences in gene mutation spectrum between ITH-H and ITH-L groups. The genes with the highest mutation frequency in ITH-H group were TP53 (57.14%), DNAH5 (38.1%), TNRC18 (38.1%), TTN (38.1%), CSMD3 (33.33%), MDN1 (33.33%), MRPL49 (33.33%), and MUC19 (33.33%), while the genes with the highest mutation frequency in ITH-L group were KDM6A (83.33%), TP53 (66.67%), CALN1 (50%), MICAL2 (50%), MTOR (50%), NINL (50%), PCDHA (50%), and RMIBP2 (50%). The objective response rate (ORR) of immunotherapy was 66.66% and 42.86% for the patients with ITH-L and ITH-H subgroup. The ITH-L patients had significantly improved median overall survival (mOS) than the ITH-H group (not reach versus 12.3 months, hazard ratio (HR) = 6.65, 95%CI 0.86−51.36, P = 0.037). There was no significant difference in median TMB levels between ITH-L and ITH-H groups (8 Muts/Mb versus 7.6 Muts/Mb, P = 0.97). The TMB high (TMB-H) patients had no significantly better mOS than the TMB low (TMB-L) group (not reach versus 9.07 months, HR = 0.33, 95%CI 0.07−1.49, P = 0.13). A pooled analysis of ITH and TMB show that mOS in the ITH-L+ TMB-H group was numerically better than that in the ITH-H+TMB-H, ITH-H+TMB-L, and ITH-L+TMB-L group (not reach versus 15.6 months versus 7.1 months versus not reach, HR-1.57, 95CI 0−Inf, P = 0.1). Conclusions: The results suggest that ITH may be independent of TMB and a positive predictor of immunotherapy in patients with advanced BLCA.

Analysis of ATM germline mutations in solid tumors from Chinese patients.

10586 Background: Ataxia-telangiectasia mutated (ATM) is a tumor suppressor genes and plays an important role in the repair of DNA double-strand breaks (DSBs). Germ-line mutations of ATM gene result in the well-characterized ataxia telangiectasia syndrome, which manifests with an increased cancer predisposition, including a 20% to 30% lifetime risk of lymphoid, gastric, breast, central nervous system, skin, and other cancers. Novel therapies are in development that may improve the response to therapy of patients with ATM-deficient cancers including targeted therapy and immunotherapy. Nevertheless, the knowledge of incidence of ATM germline mutations in solid tumor remains poorly understood. Methods: We identified 19248 malignant solid tumors patients without selecting age or family history in a retrospective cohort. The pathogenicity of germline mutations was categorized based on American College of Medical Genetics and Genomics (ACMG) guidelines.The patients were divided into two groups, P/ LP group (with pathogenic or likely-pathogenic mutations), and Non-P/LP group (neither pathogenic nor likely-pathogenic mutation). Results: In 19248 patients with pan-cancer, 76 patients were found 76 heterozygous pathogenic or likely pathogenic germline mutations in ATM gene and the mutation frequency was 0.39%.Among these 76 carriers, 71 (93.4%) carried frameshift, nonsense or splice mutations (33 for frameshift,21 for missense and 17 for splice mutations). 4 carried missense mutations and 1 carried intron mutations. Eliminated a few types of cancers that had a smaller number (less than 50), the higher frequency of mutations contained cancers were pancreatic cancer (1.0%,7/692), breast cancer (0.71%,3/423), gastric cancer (0.6%,7/1094), The median age of group P/LP and non-P/LP group was 64 and 63 in pancreatic cancer，62 and 53 in breast cancer and 62 and 61 in gastric cancer. The three genes with the most somatic mutations were TP53, MUC16 and KRAS in P/LP group and were TP53, MUC16 and EGFR in non-P/LP group. There was no significant difference in mutation frequency of the four genes. The media TMB in P/LP group and non-P/LP group were 3.55 and 2.84 respectively and there was no statistical difference. In P/LP group，only one patients is high microsatellite instability who carryied a MLH1 and a ATM pathogenic mutation. Conclusions: This was the first report of the incidence of ATM germline mutations in Chinese solid tumors which expanded the understanding of ATM and provided a direction for clinical trial design of novel therapies. The relationship between germline mutations and cancer susceptibility will be studied in the future.

Clinical and genomic characteristics between DDR germline and somatic mutations in pan-cancer.

e15139 Background: The defects DNA-damage repair (DDR) genes would drive tumor formation and are associated with increased genomic instability and tumor mutational burden (TMB) in cancer. Although, alterations of DDR genes are common in tumor, the differences between the germline and somatic alterations are poorly characterized. Methods: The DNA sequencing data of 540 genes from 6174 pan-cancer patients were retrospectively collected and 276 DDR genes were analyzed. The variations were annotated as pathogenic (P), likely pathogenic (LP), and nonpathogenic (NP) according to ACMG (American College of Medical Genetics) guideline. The patients were divided into 3 groups: DDR-germline (P&LP germline variants, N = 766), DDR-somatic group (only P&LP somatic variants, N = 2923) and non-DDR group (NP variants, N = 2485). Results: In pan-cancer, 12.41% patients have germline DDR gene mutations, of which tumors with high germline mutation frequency include ovarian cancer (27/102, 26.47%), glioma (22/136, 16.18%), breast cancer (25/159, 15.72%), genitourinary cancer (17/114, 14.91%), biliary tract cancer (42/284, 14.79%), pancreatic cancer (28/193, 14.51%), colorectal cancer (95/727, 13.07%), head-neck tumors (11/85, 12.94%) and esophagus cancer (16/124, 12.90%). Meanwhile, 47.34% patients have somatic DDR gene mutations, of which tumors with high somatic mutation frequency include esophagus cancer (89/124, 71.77%), colorectal cancer (488/727, 67.13%), endometrial cancer (49/73, 67.12%), head-neck tumors (50/85, 58.82%), pancreatic cancer (111/193, 57.51%), gastric carcinoma (233/415, 56.14%), ovarian cancer (53/102, 51.96%) and biliary tract cancer (138/284, 48.59%). The most commonly germline alterations were found in TP53 (33%), PTEN (14%) and BRCA2 (14%), while in the somatic mutations, TP53 (52%) showed the highest frequency, PTEN (9%) and MSH3 (8%) followed. In pairwise comparisons of the three groups’ actionable mutations, DDR-somatic group was more likely to have alterations in TP53 (OR = 5.68), AKT3 (OR = 5.81) and APC (OR = 1.42) compared with DDR-germline group. Taken non-DDR group as reference, mutations in TP53 (OR = 5.09), PTEN (OR = 7.65), BRCA2 (OR = 5.32) were more common in DDR-germline group, but TP53 (OR = 28.90), PTEN (OR = 7.73), APC (OR = 2.68) in DDR-somatic group. The tumor mutational burden (TMB) was statistically different among the three groups (p < 2.22e−16). The DDR-somatic group exhibits the highest median TMB (5.68) compared with 1.42 in DDR-germline and 2.13 in non-DDR group. Conclusions: The differences in the mutation profile between the DDR-germline and DDR-somatic groups and the distinct actionable genes may indicate the different target-therapy choices. Besides, DDR-somatic group exhibited the highest TMB, which might indicate patients with somatic DDR alterations may better benefit from the immunotherapy.

PROGNOSTIC SIGNIFICANCE OF KAT2A AS A HISTONE ACETYLATION REGULATOR IN DIFFUSE LARGE B‐CELL LYMPHOMA

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphoid neoplasm with variations in the expression of genes and genetic alterations. Histone acetylation is one of the epigenetic alterations associated with lymphoma pathogenesis. Among the many types of cell death, ferroptosis has distinct properties and has been found to be associated with physical conditions as well as various cancers. Using consensus clustering, we categorized DLBCL patients based on histone acetylation regulators and ferroptosis-related genes (HFGs) expression. LASSO Cox regression was used to calculate risk scores. Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis, and ROC curve analysis were performed. qRT-PCR was used to validate the expression of four HFGs in DLBCL cell lines. According to the study, CREBBP had the highest mutation frequency. Based on CREBBP mutations, patients were divided into mutant and wild groups, and several histone acetylation regulators were overexpressed in the mutant group. To demonstrate the potential malignant mechanisms of HFGs in DLBCL development and their prognostic value. A significant positive self-correlation was observed in HAGs and FRGs. A negative correlation was found between KAT2A, the HA writer, and the majority of FRGs (Fig. 1A). 36 genes were selected (p < 0.05) by univariate regression correlations with clinical prognosis. Consensus clustering was used to categorize the expression of DLBCL patients. K-M survival analysis showed that HFcluster B had a worse prognosis. Based on HFGs, we developed a high-quality and high-accuracy gene signature prediction model. Following LASSO Cox regression, a four-gene signature model was identified. Patients with DLBCL in the high-risk group had a significantly shorter OS (p < 0.001, Figure 1B). ROC curves, univariate and multivariate Cox regression analyses also revealed that riskScore served as an independent indicator for patients with DLBCL (p < 0.001). Analyses revealed that molecular mechanisms mediate histone acetylation and ferroptosis. The results of PCR in CD19+ B cells and DLBCL cell lines also confirmed the accuracy of the model. Due to its close relationship to ferroptosis-related genes, KAT2A was explored in greater depth. Further study revealed that KAT2A was significantly correlated with age (p = 0.028) and IPI (p = 0.016). In univariate analysis, KAT2A was an independent indicator of DLBCL patients (p < 0.001). The expression level of KAT2A was negatively correlated with OS in DLBCL patients (p = 0.003). Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Homologous recombination-related (HRR) gene mutations in patients with glioma and their relevance to genomic characteristics, tumor mutation burden (TMB), and prognosis.

e14023 Background: Homologous Recombination Repair (HRR) is one of the core repair methods for DNA double strand damage and the mutations of HRR pathway gene may be the main cause of many cancers. However, there is little research on the potential correlation between the molecular characteristics of HRR pathway genes and glioma. Our study explored HRR gene mutations in glioma patients and its relevance to genomic characteristics, tumor mutation burden (TMB) and prognosis. Methods: Samples from 367 glioma patients with a median age of 46 were detected by 551-gene panel or whole-exome sequencing (WES), and the WES data of 894 glioma patients were extracted from TCGA database ( http://cancergenome.nih.gov ). Among 367 glioma patients, 151 patients occurred HRR somatic mutations. Among them, 47.68% (72/151) patients carried HRR related pathogenic mutation (P/LP), and 52.32% (79/151) patients carried HRR benign/likely benign/variant of uncertain significance mutations (B/LB/VUS). The patients were divided into two groups according to whether patients carried HRR related pathogenic mutation, HRR P/LP group (72) and Non-HRR P/LP group (295). The differences of genomic characteristics, TMB and prognosis between HRR P/LP group and Non-HRR P/LP group were analyzed. Results: In 151 glioma patients harboring HRR somatic mutations, PTEN had the highest mutation frequency (49%; 74/151), followed by CHEK2, DNMT3A, BRCA2 and ATM, with mutation frequency of 11% (17/151), 10% (15/151), 9% (14/151）and 9% (14/151). Patients of HRR P/LP group and HRR B/LB/UVS group showed a significant difference at HRR pathway genes, including PTEN, RAD50, MSH6 and RAD21. In addition, differences of non-HRR pathway genes in HRR P/LP group and Non-HRR P/LP group were found. The mutation frequency of these differential genes was generally high in HRR P/LP group. However, compared to Non-HRR P/LP group, the mutation frequency of IDH1 in HRR P/LP group was significantly higher (Non-HRR P/LP 32% VS HRR P/LP 10%; P＜0.0001). The TMB value in Non-HRR P/LP group was significantly higher than that in HRR P/LP group (P＜0.0001), and the same trend of TMB in two groups was discovered in TCGA cohort (P＜0.0001). Importantly, a significant difference of overall survival (OS) in TCGA database was found between HRR P/LP group and Non-HRR P/LP group (P＜0.0001, HR =2.17, 95%CI: 1.54-3.06), and the median OS were 17.6 and 34.9 months, respectively. Conclusions: This study showed HRR pathway genes status was significantly related to the genomic characteristics, TMB and prognosis of glioma patients. Compared to patients in HRR P/LP group, patients in Non-HRR P/LP group had higher mutation frequency of IDH1, lower TMB value and better prognosis. The relationship between HRR pathway genes and glioma needs to be further explored.

High frequency of germline mutations in DDR associated genes SLX4, MSH6, CDK4, TSC2, and BRCA2 might contribute to the occurrence of MPLC.

e20514 Background: Multiple primary lung cancer (MPLC) is the presence of two or more distinct and separate lung tumors in a single patient. Each is considered a primary tumor rather than a metastasis from another site. This phenomenon has been the subject of several studies in recent years due to its increasing incidence and potential implications for patient management and prognosis. The underlying causes of MPLCs are not fully understood. Still, several factors have been proposed to contribute, including the genetic predisposition of carcinogens, such as those in the TP53, BRCA1, and BRCA2 genes, which are associated with an increased risk of developing MPLCs, as well as other cancers. Whether the presence of germline genetic mutations can increase an individual's susceptibility and contribute to the development of MPLCs is not clear yet. Here we reported the landscape of germline mutations of 77 MPLC patients. Methods: We retrospectively analyzed the somatic and germline mutations from 77 MPLC patients by NGS analysis. The GO and KEGG analyses were performed based on the top 30 most frequent germline mutations. Results: Among the 77 patients, 21 (27.27%) were male, and 56 (72.73%) were female. The median age was 60 (26-82) years old. The top 5 frequent germline mutated genes were SLX4 (34.92%), MSH6 (39.68%), CDK4 (26.98%), TSC2 (25.4%), and BRCA2 (25.4%). The top 5 frequent somatic mutated genes were EGFR (80.95%), CDK4 (26.98%), MCL1 (23.81%), SOCS1 (19.05%), and TP53 (19.05%). GO analysis of germline mutations revealed enrichment in ontologies such as response to radiation and UV, cell cycle regulation, a series of DNA damage repair associated ontologies, and telomere organization. KEGG analysis indicated that these germline mutations were enriched in the PI3K-Akt pathway, microRNAs in cancer, TP53 pathway, Fanconi anemia pathway, cellular senescence, and many other cancer-associated pathways. Conclusions: Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. In our analysis, the MPLC patients contained widespread germline mutations in SLX4, MSH6, CDK4, TSC2, and BRCA2, which were all associated with DNA damage repair. Besides, GO analysis and KEGG analysis also revealed enrichment in DDR-associated ontologies or pathways (e.g., TP53 pathway, Fanconi anemia pathway, cellular senescence pathways). To our knowledge, an underlying hallmark of cancers is their genomic instability, which is associated with a greater propensity to accumulate DNA damage. The widespread of germline mutations in DDR-associated genes, including SLX4, MSH6, CDK4, TSC2, and BRCA2, might contribute to the gene instability and promote the development of MPLC.

Genomic and molecular landscape of homologous recombination deficiency across multiple cancer types

Homologous recombination deficiency (HRD) causes faulty double-strand break repair and is a prevalent cause of tumorigenesis. However, the incidence of HRD and its clinical significance in pan-cancer patients remain unknown. Using computational analysis of Single-nucleotide polymorphism array data from 10,619 cancer patients, we demonstrate that HRD frequently occurs across multiple cancer types. Analysis of the pan-cancer cohort revealed that HRD is not only a biomarker for ovarian cancer and triple-negative breast cancer, but also has clinical prognostic value in numerous cancer types, including adrenocortical cancer and thymoma. We discovered that homologous recombination–related genes have a high mutation or deletion frequency. Pathway analysis shows HRD is positively correlated with the DNA damage response and the immune-related signaling pathways. Single cell RNA sequencing of tumor-infiltrating lymphocytes reveals a significantly higher proportion of exhausted T cells in HRD patients, indicating pre-existing immunity. Finally, HRD could be utilized to predict pan-cancer patients’ responses to Programmed cell death protein 1 immunotherapy. In summary, our work establishes a comprehensive map of HRD in pan-cancer. The findings have significant implications for expanding the scope of Poly ADP-ribose polymerase inhibitor therapy and, possibly, immunotherapy.

Spectrum of MECP2 mutations in Iranian Azeri Turkish Rett syndrome patients

Rett syndrome is an X-linked dominant neurodevelopmental disorder that occurs mostly in females. De novo mutations in the MECP2 gene have an important role in the appearance of the features of this syndrome. We planned to study spectrum of MECP2 mutations in Rett syndrome patients and their clinical symptoms. A cohort of 29 patients referred by neurologists from Iranian Azeri Turks was screened. Then direct sequencing was utilized to characterize the spectrum of mutations in the MECP2 gene in Rett syndrome patients. A total of 10 different mutations on MECP2 gene were detected in 22 patients. We identified 2 (9%) frameshift, 10 (45.64%) nonsense, 8 (36.4%) missense mutations, and 2(9%) large deletions. In this cohort, one of the detected deletions was novel, namely 1023-1096del74nt. Random X chromosome inactivation in females’ cells and different MECP2 mutations can cause a phenotypic variability between patients. This is the first report regarding the spectrum of MECP2 mutation and phenotypic spectrum in Iranian Azeri Turks with Rett syndrome. Our finding confirms a high mutation frequency (75.8%) of MECP2 gene in Iranian Rett syndrome patients.

Genomic characteristics and prognosis of lung cancer patients with MSI-H: A cohort study

BackgroundMicrosatellite instability (MSI) is the first pan-cancer biomarker approved to guide immune checkpoint inhibitor therapy for MSI-high (MSI-H) solid tumors. In lung cancer, the MSI-H frequency is very low, and the genetic characteristics and prognosis of lung cancer with MSI-H were rarely reported. MethodsNext-generation sequencing and immunohistochemistry were used detect MSI status, tumor mutation burden (TMB) and PD-L1 expression. ResultsAmong 12,484 lung cancer patients screened, 66 were found with MSI-H, the proportion was as low as 0.5%. Compared with Microsatellite stability (MSS), TMB was higher in MSI-H lung cancer patients, while PD-L1 expression showed no considerable difference between MSI-H and MSS. After propensity score matching, compared with MSS, the most common companion mutations in MSI-H were TP53, BRCA2, TGFBR2, PTEN and KMT2C. In MSI-H lung adenocarcinoma with EGFR mutation, TGFBR2 and ERBB2 had higher mutation frequency than in MSS. ConclusionThe current study reveals the genetic characteristics of MSI-H lung cancer, which advanced our understanding of MSI-H lung cancer.

Impact of mini-driver genes in the prognosis and tumor features of colorectal cancer samples: a novel perspective to support current biomarkers.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, and its development is associated with the gains and/or losses of genetic material, which leads to the emergence of main driver genes with higher mutational frequency. In addition, there are other genes with mutations that have weak tumor-promoting effects, known as mini-drivers, which could aggravate the development of oncogenesis when they occur together. The aim of our work was to use computer analysis to explore the survival impact, frequency, and incidence of mutations of possible mini-driver genes to be used for the prognosis of CRC. We retrieved data from three sources of CRC samples using the cBioPortal platform and analyzed the mutational frequency to exclude genes with driver features and those mutated in less than 5% of the original cohort. We also observed that the mutational profile of these mini-driver candidates is associated with variations in the expression levels. The candidate genes obtained were subjected to Kaplan-Meier curve analysis, making a comparison between mutated and wild-type samples for each gene using a p-value threshold of 0.01. After gene filtering by mutational frequency, we obtained 159 genes of which 60 were associated with a high accumulation of total somatic mutations with Log2 (fold change) > 2 and p values < 10-5. In addition, these genes were enriched to oncogenic pathways such as epithelium-mesenchymal transition, hsa-miR-218-5p downregulation, and extracellular matrix organization. Our analysis identified five genes with possible implications as mini-drivers: DOCK3, FN1, PAPPA2, DNAH11, and FBN2. Furthermore, we evaluated a combined classification where CRC patients with at least one mutation in any of these genes were separated from the main cohort obtaining a p-value < 0.001 in the evaluation of CRC prognosis. Our study suggests that the identification and incorporation of mini-driver genes in addition to known driver genes could enhance the accuracy of prognostic biomarkers for CRC.

High carrier frequency of pathogenic PATL2 gene mutations predicted in population: a bioinformatics-based approach.

Topoisomerase II homologue 2 (PATL2) has been confirmed to be a key gene that contributes to oocyte maturation. However, the allele distribution and carrier frequency of these mutations remain uncharacterized. So a bioinformatics subcategory analysis of PATL2 mutations from outcome data and Single Nucleotide Polymorphism (SNP) databases was conducted. Altogether, the causative PATL2 mutation number detected in patients with oocyte maturation defects in the clinical studies and pathogenic PATL2 mutation sites predicted by software based on the database was approximately 53. The estimated carrier frequency of pathogenic mutation sites was at least 1.14‰ based on the gnomAD and ExAC database, which was approximately 1/877. The highest frequency of mutations detected in the independent patients was c.223-14_223-2del13. The carrier frequency of this mutation in the population was 0.25‰, which may be a potential threat to fertility. Estimated allele and carrier frequency are relatively higher than those predicted previously based on clinical ascertainment. A review of PATL2 mutation lineage identified in 34 patients showed that 53.81%, 9.22% and 14.72% of the oocytes with PATL2 mutations were arrested at the germinal vesicle (GV) stage, metaphase I (MI) stage and first polar body stage, respectively. Oocytes that could develop to the first polar body stage were extremely rare to fertilise, and their ultimate fate was early embryonic arrest. Phenotypic variability is related to the function of the regions and degree of loss of function of PATL2 protein. A 3D protein structure changes predicted by online tools, AlphaFold, showed aberrations at the mutation sites, which may explain partially the function loss. When the mutated and wild-type proteins are not in the same amino acid category, the protein structure will be considerably unstable. The integration of additional mutation sites with phenotypes is helpful in drawing a complete picture of the disease. Bioinformatics analysis of PATL2 mutations will help reveal molecular epidemiological characteristics and provide an important reference for new mutation assessment, genetic counselling and drug research.

Aberrant expression of NPPB through YAP1 and TAZ activation in mesothelioma with Hippo pathway gene alterations.

Mesothelioma is a neoplastic disease associated with asbestos exposure. It is highly malignant and has a poor prognosis; thus, early detection is desirable. Recent whole-genome analysis has revealed that mesothelioma is characterized by a high frequency of mutations in a set of genes involved in the Hippo pathway, such as NF2 and LATS2. However, a rapid, simple, and precise method for finding mesothelioma with these mutations has not yet been established. Clustering of Hippo pathway gene alteration groups and the differential expression of each gene in mesothelioma patients were analyzed using The Cancer Genome Atlas database. Gene expression levels in various tumors and normal tissues were analyzed using public databases. Knockdown or transient expression of YAP1 or TAZ was performed to evaluate the regulation of gene expression by these genes. NT-proBNP was measured in the pleural effusions of 18 patients and was compared with NF2 expression in five cases where cell lines had been successfully established. NPPB mRNA expression was markedly higher in the group of mesothelioma patients with Hippo pathway gene mutations than in the group without them. NPPB expression was low in all normal tissues except heart, and was highest in mesothelioma. Mesothelioma patients in the high NPPB expression group had a significantly worse prognosis than those in the low NPPB expression group. NPPB expression was suppressed by knockdown of YAP1 or TAZ. NT-proBNP was abundant in the effusions of mesothelioma patients and was particularly high in those with impaired NF2 expression. NPPB, whose levels can be measured in pleural effusions of mesothelioma patients, has the potential to act as a biomarker to detect NF2-Hippo pathway gene alterations and/or predict patient prognosis. Additionally, it may provide useful molecular insights for a better understanding of mesothelioma pathogenesis and for the development of novel therapies.

Cuproptosis-Related Genes CDK1 and COA6 Involved in the Prognosis Prediction of Liver Hepatocellular Carcinoma.

Liver hepatocellular carcinoma (LIHC) is the most frequently seen type of primary liver cancer. Cuproptosis is a novel form of cell death highly associated with mitochondrial metabolism. However, the clinical impact and pertinent mechanism of cuproptosis genes in LIHC remain largely unknown. From public databases, we systematically assessed common genes from LIHC differentially expressed genes (DEGs) and cuproptosis-related genes using bioinformatics analysis. These common genes were then analyzed by enrichment analysis, mutation analysis, risk score model, and others to find candidate hub genes related to LIHC and cuproptosis. Next, hub genes were determined by expression, clinical factors, immunoassay, and prognostic nomogram. Based on 129 cuproptosis-related genes and 3492 LIHC DEGs, we totally identified 21 downregulated and 18 upregulated common genes, and they were enriched in pathways, such as zinc ion homeostasis and oxidative phosphorylation. In the mutation analysis, missense mutation was the most common type in LIHC patients, and the common gene F5 had the highest mutation frequency. After LASSO-Cox regression analysis and prognostic analysis, CDK1, ABCB6, LCAT, and COA6 were identified as prognostic signature genes. Among them, ABCB6 and LCAT were lowly expressed in tumors, and CDK1 and COA6 were highly expressed in tumors. In addition, ABCB6 and LCAT were negatively correlated with 6 kinds of immune cells, while CDK1 and COA6 were positively correlated with them. CDK1 and COA6 were identified as hub genes related to LIHC by Cox regression analysis and prognostic nomogram. CDK1 and COA6 are two oncogenes in LIHC, which are involved in the molecular mechanism of cuproptosis and LIHC. Besides, CDK1 and COA6 can positively regulate the expressions of immune cells in LIHC. In clinical practice, they can be used as immunotherapeutic targets and prognostic predictors in LIHC, which sheds new light on the scientific fields of cuproptosis and LIHC.

Multi-omics analysis of Siglec family genes in cutaneous melanoma.

Melanoma is widely recognized as the most aggressive and fatal type of skin cancer; however, effective prognostic markers are lacking. The sialic acid-binding immunoglobulin-type lectin (Siglec) gene family plays an important role in the development of tumors and immune escape, but its prognostic role in melanoma remains unknown. Siglec genes have a high mutation frequency, with up to 8% in SIGLEC7. High expression levels of Siglecs in tumor bulk suggests a better prognosis. Siglecs also show a high degree of synergistic expression. Immunohistochemistry was used to analyze the expression of SIGLEC9 in tumor tissue microarray. The expression of SIGLEC9 in tumor tissue without metastasis was higher than that in tumor tissue with metastasis. We used unsupervised clustering to create a high expression of Siglec (HES) cluster and a low expression of Siglec (LES) cluster. The HES cluster correlated with high overall survival and increased expression levels of Siglec genes. The HES cluster also showed significant immune cell infiltration and activation of immune signaling pathways. We used least absolute shrinkage and selection operator (LASSO) regression analysis to reduce the dimensionality of Siglec cluster-related genes and constructed a prognostic model composed of SRGN and GBP4, which can risk-stratify patients in both the training and test datasets. We conducted a multi-omics analysis of the Siglec family genes in melanoma and found that Siglecs play an important role in the occurrence and development of melanoma. Typing constructed using Siglecs can show risk stratification and derived prognostic models can predict a patient's risk score. In summary, Siglec family genes are potential targets for melanoma treatment as well as prognostic markers that can direct individualized treatments and improve overall survival.

Epidermal Growth Factor Receptor Mutation Status and the Impact on Clinical Outcomes in Patients with Non-Small Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) mutation status differs according to ethnicity, gender, smoking history, and histology types. The present study aimed to evaluate EGFR mutation status in patients with non-small cell lung cancer (NSCLC) and further explore its association with clinical characteristics and prognosis in advanced NSCLC patients (Stage IIIB-IV). 238 NSCLC patients were enrolled in this study from October 2016 through December 2019. Patient characteristics and clinical data including age, gender, smoking history, histology types, tumor stage, survival status, and time were collected via electronic medical record system or telephone. 21 somatic mutations which spanned exons 18-21 of EGFR were detected using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, followed by analysis of links to clinical characteristics, progression-free survival (PFS) and overall survival (OS). 103 patients were detected harboring EGFR mutations among the 238 cases tested (43.3%), and exons 19 and 21 were the highest mutation frequencies, with 20.6% and 19.3% respectively. The EGFR mutation rate was much higher in female versus male (57.4% vs 31.5%, p <0.001), in non-smokers compared to smokers (56.8% vs 25.9%, p <0.001), and in those with adenocarcinoma than other histology types (48.3% vs 3.7%, p <0.001). For patients in advanced stage, median PFS was 11 months in patients harboring EGFR mutations, versus 4 months in patients with wild type EGFR (p <0.001); median OS was 24 versus 12 months (p <0.001). Never smoking (p = 0.042) and adenocarcinoma (p = 0.007) were independent favorable factors for EGFR mutations. Our data strengthen the findings of high prevalence of EGFR mutations in Asian patients with NSCLC. Mutations are prevalent in those patients who are female, adenocarcinoma, and have never smoked. Moreover, advanced EGFR mutation-positive patients have better PFS and OS than those with wild type EGFR.

Comprehensive genetic analyses of childhood acute leukemia in Iraq using next-generation sequencing.

Molecular analyses in hematological malignancies provide insights about genetic makeup. Probable etiological factors in leukemogenesis could also be disclosed. Since genetic analyses are still primitive in Iraq, a country of repeated wars, we conceived of performing next-generation sequencing (NGS), to disclose the genomic landscape of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) among a cohort of Iraqi children. Dried blood samples were collected from Iraqi children with ALL (n=55), or AML (n=11), and transferred to Japan where NGS was done. Whole-exome, whole-genome, and targeted gene sequencings were performed. Somatic point mutations and the copy number variations among Iraqi children with acute leukemia were comparable with those in other countries, and cytosine-to-thymine nucleotide alterations were dominant. Strikingly, TCF3-PBX1 was the most recurrent fusion gene (22.4%) in B-cell precursor ALL (B-ALL), and acute promyelocytic leukemia (AML-M3) was subtyped in 5 AML cases. Additionally, a high frequency of RAS signaling pathway mutations was detected in children with B-ALL (38.8%), along with 3 AML cases that carried oncogenic RAS. Apart from disclosing the high frequency of TCF3-PBX1, NGS confirmed our previous finding of recurrent RAS mutations in Iraqi childhood acute leukemia. Our results suggest that the biology of Iraqi childhood acute leukemia is in part characteristic, where the war-aftermath environment or geography might play a role.

Extensive intratumor regional epigenetic heterogeneity in clear cell renal cell carcinoma targets kidney enhancers and is associated with poor outcome

BackgroundClear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations.ResultsA multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential.ConclusionTaken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers.

Whole-exome sequencing for screening noise-induced hearing loss susceptibility genes

Background High-throughput sequencing of genes indicating susceptibility to noise-induced hearing loss has not previously been reported. Aims/Objectives To identify and analyze genes associated with susceptibility to noise-induced hearing loss (NIHL) and characterize differences in susceptibility to hearing loss by genotype. Material and methods Pure tone audiometry tests were performed on 113 workers exposed to high-intensity noise. Whole-exome sequencing (WES) was conducted and NIHL susceptibility genes screened for training unsupervised and supervised machine learning models. Immunofluorescence staining of mouse cochlea was used to observe patterns of NIHL susceptibility gene expression. Results Participants were divided into a NIHL and a control group, according to the results of audiometry tests. Seventy-three possible NIHL susceptibility genes were input into the machine learning model. Two subgroups of NIHL could be distinguished by unsupervised machine learning and the classification was evaluated by the supervised machine learning algorithm. The VWF gene had the highest mutation frequency in the NIHL group and was expressed mainly in the spiral ligament. Conclusions and significance NIHL susceptibility genes were screened and NIHL subgroups could be distinguished. VWF may be a novel NIHL susceptibility gene.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain–in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Exploration of the immunogenetic landscape of hyperprogressive disease after combined immunotherapy in cancer patients

The immune-genetic changes that occur in cancer patients experiencing hyperprogressive disease (HPD) during combined immunotherapy are unclear. In thisstudy, HPD patients with pre- and post-HPD samples and non-HPD patients with solid tumors were molecularly characterized by genetic and tumor immune microenvironment (TiME) analyses of paired samples by whole-exome sequencing, RNA sequencing, and multiplex immunofluorescence. The genetic analysis of paired samples showed that almost all the tumor driver gene mutations were preserved between pre- and post-HPD tumors. HPD patients had higher frequencies of mutations in TP53 and CNN2, and a significantly higher mutant-allele tumor heterogeneity than non-HPD patients. Tumor IL-6 mRNA was upregulated in post-HPD samples vs. pre-HPD, accompanied by a potential immune suppressive TiME with an elevated M2/M1 ratio. Salvage treatment with irinotecan plus bevacizumab was effective in one HPD patient, who experienced prolonged survival. These genetic features and TiME characteristics might help identify the features of HPD after immunotherapy.