Genomic Features of Interstitial Deletions of Chromosome 9q in Acute Myeloid Leukemia.

Interstitial deletion in the long arm of chromosome 9 [del(9q)] is a fairly common cytogenetic finding associated with acute myeloid leukemia (AML), seen in approximately 2-5% of AML patients. However, the genomic features of the deletion remain largely unknown. Using chromosome analysis, single nucleotide polymorphism microarray, and next-generation sequencing, we characterized del(9q)s and other genomic alterations in 9 AML patients. We found several distinct features of the del(9q)s. The proximal breakpoints of the deletions are clustered within a 2.5-Mb region (chr9: 68,513,625-70,984,372; GRCh37) enriched with segmental duplications, which may represent a "hotspot" for genomic rearrangements. However, the distal breakpoints of the deletions vary significantly. In addition, the overall deleted region could be divided into a 14.4-Mb proximal constitutional region (chr9: 70,950,015-85,397,699; 9q21.11q21.32) and a 24.0-Mb distal oncogenic region (chr9: 85,397,700-109,427,261; 9q21.32q31.1). We further identified a 6.8-Mb common overlapped deletion region (CODR) in the distal region (chr9: 90,590,650-97,366,400). This CODR carries multiple genes that are reportedly involved in cancer pathogenesis. The prognostic value of the del(9q) in AML apparently depends on additional genomic alterations in the patients.

Frequency of pathogenic/likely pathogenic germline variants in cancer-related genes among children with acute leukemia in Saudi Arabia.

BACKGROUND: The frequency of pathogenic/likely pathogenic (P/LP) germline mutations in cancer-related genes among children with cancer in highly consanguineous populations is not well studied. METHODS: Whole-exome sequencing of germline DNA was performed in 60 children with acute leukemia. We used the St. Jude Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE) data portal for the classification of germline variants by the St. Jude Medal Ceremony pipeline. RESULTS: Fifty-seven patients had acute lymphoblastic leukemia (ALL) and three patients had acute myeloid leukemia. Parental consanguinity was present in 27 (45%) patients. All patients were of Arab ancestry. Three patients (5%) had a history of cancer in their siblings. Five patients (8.3%) had P/LP germline mutations in cancer-related genes. Three patients with B-ALL had heterozygous pathogenic mutations in TP53, BRCA1, and BRCA2; one patient with B-ALL had homozygous pathogenic mutation in PMS2; and one patient with T-ALL had LP homozygous mutation in AK2 that was associated with reticular dysgenesis. Among patients who had history of parental consanguinity, three (11%) had P/LP germline mutations compared with two (8%) in the absence of parental consanguinity. Fourteen (23%) patients had gold medal variants in cancer-related genes, 13 were heterozygous, and one was homozygous. Silver medal variants were present in 35 (58%) patients; all were heterozygous except one homozygous. CONCLUSIONS: Children with acute leukemia in Saudi Arabia had low frequency of P/LP mutations in cancer-related genes despite the high rate of consanguinity. Larger studies using whole-genome sequencing are needed to further explore the heritability of childhood leukemia.

Nonsynonymous, synonymous and nonsense mutations in human cancer-related genes undergo stronger purifying selections than expectation.

BACKGROUND: Nonsynonymous mutations change the protein sequences and are frequently subjected to natural selection. The same goes for nonsense mutations that introduce pre-mature stop codons into CDSs (coding sequences). Synonymous mutations, however, are intuitively thought to be functionally silent and evolutionarily neutral. Now researchers know that the optimized synonymous codon usage is advantageous in the speedy mRNA translation process. With the advent of NGS technique, the explosion of NGS data generated from the tumor tissues help researchers identify driver mutations in cancer-related genes, but relatively less attention is paid to the SNP data in healthy human populations when studying cancer. METHODS: Here, we analyzed the publically available human SNPs. We classified these SNPs according to their functional and evolutionary categories. By simply dividing the human genes into cancer-related genes and other genes, we compared the features of nonsynonymous, synonymous and nonsense mutations in these two gene sets from multiple aspects. RESULTS: We provided lines of evidence that the nonsynonymous, synonymous and nonsense mutations in cancer-related genes undergo stronger purifying selection when compared to the expected pattern in other genes. The lower nonsynonymous to synonymous ratio observed in cancer-related genes suggests the suppression of amino acid substitutions in these genes. The synonymous SNPs, after excluding those in splicing regions, exhibit preferred changes in codon usage and higher codon frequencies in cancer-related genes compared to other genes, indicating the constraint exerted on these mutations. Nonsense mutations are less frequent and located closer to stop codons in cancer-related genes than in other genes, which putatively minimize their deleterious effects. CONCLUSION: Our study demonstrated the evolutionary constraint on mutations in CDS of cancer-related genes without the requirement of data from cancer tissues or patients. Our work provides novel perspectives on interpreting the constraint on mutations in cancer-related genes. We reveal extra constraint on synonymous mutations in cancer-related genes which is related to codon usage bias and is in addition to the splicing effect.

MGOGP: a gene module-based heuristic algorithm for cancer-related gene prioritization.

BACKGROUND: Prioritizing genes according to their associations with a cancer allows researchers to explore genes in more informed ways. By far, Gene-centric or network-centric gene prioritization methods are predominated. Genes and their protein products carry out cellular processes in the context of functional modules. Dysfunctional gene modules have been previously reported to have associations with cancer. However, gene module information has seldom been considered in cancer-related gene prioritization. RESULTS: In this study, we propose a novel method, MGOGP (Module and Gene Ontology-based Gene Prioritization), for cancer-related gene prioritization. Different from other methods, MGOGP ranks genes considering information of both individual genes and their affiliated modules, and utilize Gene Ontology (GO) based fuzzy measure value as well as known cancer-related genes as heuristics. The performance of the proposed method is comprehensively validated by using both breast cancer and prostate cancer datasets, and by comparison with other methods. Results show that MGOGP outperforms other methods, and successfully prioritizes more genes with literature confirmed evidence. CONCLUSIONS: This work will aid researchers in the understanding of the genetic architecture of complex diseases, and improve the accuracy of diagnosis and the effectiveness of therapy.

PCA3 long noncoding RNA modulates the expression of key cancer-related genes in LNCaP prostate cancer cells.

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples.

BACKGROUND: Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. METHODS: To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). RESULTS: Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. CONCLUSIONS: High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

[The change of cancer-related genes expression profile in Nthy-ori-3-1 cell induced by the pesticide amitrole].

OBJECTIVE: To explore the possible mechanism of amitrole causing thyroid tumor in Nthy-ori-3-1 cell by differential expression microarray analysis. METHODS: After the Nthy-ori-3-1cells were treated with 1 ~ 100 g / m L amitrole for 24 h, and the effect of amitrole on the proliferation of the cells was detected by MTT assay. Then cells were treated with 100 g / m L amitrole for 24 h, and the differential expression microarray was tested. The microarray results was analyzed by GO analysis and pathway analysis. The microarray results were verified by real-time quantitative PCR. RESULTS: MTT results showed that amitole had no significant effect on the proliferation of Nthy-ori-3-1 cells. Microarray results showed that 90( 55 up-regulated, 35 down regulated) genes were significantly changed. GO analysis showed that 43( 37 up-regulated, 6 down-regulated) of the 90 changed genes were related to biological processes, and 42( 37 up-regulated, 5down-regulated) were related to molecular function, and 44( 38 up-regulated, 6 downregulated) were related to cell components. Pathway results showed that 44 signalingpathways were influenced by the differentially expressed genes, and 10 of them were closely related to tumor. The qRT-PCR results were consistent with microarray results. wnt5 b, arnt2 and bmp2 genes were significantly related with multiple tumor-associated pathways. CONCLUSION: Amitrole may cause thyroid tumor by multiple signaling pathways, and bmp2, arnt2 and wnt5 b may beits major target genes.

Protein tyrosine kinase 7: a hepatocellular carcinoma-related gene detected by triple-combination array.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. We developed a novel technique to identify cancer-related genes of HCC as follows: triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays, and methylation arrays. MATERIALS AND METHODS: Triple-combination array analysis was performed on one HCC sample from a 68-y-old female patient, and one candidate cancer-related gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in nine HCC cell lines and in samples from 48 HCC patients. Additionally, we evaluated gene expression by immunohistochemistry and Western blotting. RESULTS: Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate cancer-related gene. PTK7 was revealed to be hypermethylated (methylation value 0.826, range 0-1.0) in cancer tissue, compared with that of adjacent noncancerous tissues (0.047) by methylation array. Of the 48 clinical samples, 30 HCC samples (62.5%) showed PTK7 promoter hypermethylation. Downregulation of PTK7 (expressions in tumor tissues decreased by ≥ 50% compared with the noncancerous tissues) was significantly associated with age >60 y (P = 0.030) and elevation in serum protein induced by vitamin K absence or antagonists-II (P = 0.033). Moreover, patients with downregulation were significantly inferior in overall survival (P < 0.001) than the others. CONCLUSIONS: Our data imply that PTK7 acts as a cancer-related gene and may be a potent prognostic marker for HCC. Triple-combination array analysis was once again found to be useful in identifying cancer-related genes.

De Novo Cancer Mutations Frequently Associate with Recurrent Chromosomal Abnormalities during Long-Term Human Pluripotent Stem Cell Culture.

Human pluripotent stem cells (hPSCs) are pivotal in regenerative medicine, yet their in vitro expansion often leads to genetic abnormalities, raising concerns about their safety in clinical applications. This study analyzed ten human embryonic stem cell lines across multiple passages to elucidate the dynamics of chromosomal abnormalities and single-nucleotide variants (SNVs) in 380 cancer-related genes. Prolonged in vitro culture resulted in 80% of the lines acquiring gains of chromosome 20q or 1q, both known for conferring an in vitro growth advantage. 70% of lines also acquired other copy number variants (CNVs) outside the recurrent set. Additionally, we detected 122 SNVs in 88 genes, with all lines acquiring at least one de novo SNV during culture. Our findings showed higher loads of both CNVs and SNVs at later passages, which were due to the cumulative acquisition of mutations over a longer time in culture, and not to an increased rate of mutagenesis over time. Importantly, we observed that SNVs and rare CNVs followed the acquisition of chromosomal gains in 1q and 20q, while most of the low-passage and genetically balanced samples were devoid of cancer-associated mutations. This suggests that recurrent chromosomal abnormalities are potential drivers for the acquisition of other mutations.

Diabetes mellitus induces a novel inflammatory network involving cancer progression: Insights from bioinformatic analysis and in vitro validation.

Background: Patients with diabetes mellitus (DM) have a higher incidence of malignant tumors than people without diabetes, but the underlying molecular mechanisms are still unclear. Methods: To investigate the link between DM and cancer, we screened publicly available databases for diabetes and cancer-related genes (DCRGs) and constructed a diabetes-based cancer-associated inflammation network (DCIN). We integrated seven DCRGs into the DCIN and analyzed their role in different tumors from various perspectives. We also investigated drug sensitivity and single-cell sequencing data in colon adenocarcinoma as an example. In addition, we performed in vitro experiments to verify the expression of DCRGs and the arachidonic acid metabolic pathway. Results: Seven identified DCRGs, including PPARG, MMP9, CTNNB1, TNF, TGFB1, PTGS2, and HIF1A, were integrated to construct a DCIN. The bioinformatics analysis showed that the expression of the seven DCRGs in different tumors was significantly different, which had varied effects on diverse perspectives. Single-cell sequencing analyzed in colon cancer showed that the activity of the DCRGs was highest in Macrophage and the lowest in B cells among all cell types in adenoma and carcinoma tissue. In vitro experiments showed that the DCRGs verified by western bolt and PEG2 verified by ELISA were all highly expressed in COAD epithelial cells stimulated by high glucose. Conclusion: This study, for the first time, constructed a DCIN, which provides novel insights into the underlying mechanism of how DM increases tumor occurrence and development. Although further research is required, our results offer clues for new potential therapeutic strategies to prevent and treat malignant tumors.

Differences in integration frequencies and APOBEC3 profiles of five high-risk HPV types adheres to phylogeny.

Persistent infection with Human Papillomavirus (HPV) is responsible for almost all cases of cervical cancers, and HPV16 and HPV18 associated with the majority of these. These types differ in the proportion of viral minor nucleotide variants (MNVs) caused by APOBEC3 mutagenesis as well as integration frequencies. Whether these traits extend to other types remains uncertain. This study aimed to investigate and compare genomic variability and chromosomal integration in the two phylogenetically distinct Alpha-7 and Alpha-9 clades of carcinogenic HPV types. The TaME-seq protocol was employed to sequence cervical cell samples positive for HPV31, HPV33 or HPV45 and combine these with data from a previous study on HPV16 and HPV18. APOBEC3 mutation signatures were found in Alpha-9 (HPV16/31/33) but not in Alpha-7 (HPV18/45). HPV45 had significantly more MNVs compared to the other types. Alpha-7 had higher integration frequency compared to Alpha-9. An increase in integration frequency with increased diagnostic severity was found for Alpha-7. The results highlight important differences and broaden our understanding of the molecular mechanisms behind cervical cancer induced by high-risk HPV types from the Alpha-7 and Alpha-9 clades.

Analysis of lung cancer-related genetic changes in long-term and low-dose polyhexamethylene guanidine phosphate (PHMG-p) treated human pulmonary alveolar epithelial cells.

BACKGROUND: Lung injury elicited by respiratory exposure to humidifier disinfectants (HDs) is known as HD-associated lung injury (HDLI). Current elucidation of the molecular mechanisms related to HDLI is mostly restricted to fibrotic and inflammatory lung diseases. In our previous report, we found that lung tumors were caused by intratracheal instillation of polyhexamethylene guanidine phosphate (PHMG-p) in a rat model. However, the lung cancer-related genetic changes concomitant with the development of these lung tumors have not yet been fully defined. We aimed to discover the effect of long-term exposure of PHMG-p on normal human lung alveolar cells. METHODS: We investigated whether PHMG-p could increase distorted homeostasis of oncogenes and tumor-suppressor genes, with long-term and low-dose treatment, in human pulmonary alveolar epithelial cells (HPAEpiCs). Total RNA sequencing was performed with cells continuously treated with PHMG-p and harvested after 35 days. RESULTS: After PHMG-p treatment, genes with transcriptional expression changes of more than 2.0-fold or less than 0.5-fold were identified. Within 10 days of exposure, 2 protein-coding and 5 non-coding genes were selected, whereas in the group treated for 27-35 days, 24 protein-coding and 5 non-coding genes were identified. Furthermore, in the long-term treatment group, 11 of the 15 upregulated genes and 9 of the 14 downregulated genes were reported as oncogenes and tumor suppressor genes in lung cancer, respectively. We also found that 10 genes of the selected 24 protein-coding genes were clinically significant in lung adenocarcinoma patients. CONCLUSIONS: Our findings demonstrate that long-term exposure of human pulmonary normal alveolar cells to low-dose PHMG-p caused genetic changes, mainly in lung cancer-associated genes, in a time-dependent manner.

21-Gene Recurrence Assay Associated With Favorable Metabolic Profiles in HR-Positive, HER2-Negative Early-Stage Breast Cancer Patients.

Background: Comprehensive investigations of the associations between 21-gene recurrence assay and metabolic profiles in Chinese breast cancer patients are limited. Methods: We evaluated the relations of the 21-gene recurrence risk score (RS) and the expression of cancer-related genes with metabolic factors and biomarkers of insulin and the insulin-like growth factor (IGF) axis, and examined the interactions between the 21-gene RS and these metabolic profiles on breast cancer recurrence in Chinese women with HR-positive, HER2-negative early-stage breast cancer. Results: The 21-gene RS was inversely associated with body mass index ([BMI]ß: -0.178 kg/m2; P=0.040), the homeostasis model assessment of insulin resistance index ([HOMA-IR] ß: -0.031; P=0.042), insulin (ß: -0.036 uIU/ml; P=0.009), and C-peptide (ß: -0.021 ug/L; P=0.014) and was positively associated with high-density lipoprotein cholesterol (ß: 0.025 mmol/L; P=0.004), which were driven by the relation patterns between specific cancer-related genes and these metabolic profiles. Each 10-unit increase in the 21-gene RS was associated with 28% (95% CI: 5-47%) higher risk of breast cancer recurrence; this association was also observed in patients with favorable metabolic profiles in relevant to an absence of obesity, insulin resistance, hyperglycemia, hypertension, or dyslipidemia (28-44% higher risk) and among women with a low level of insulin, C-peptide, or the IGF1/IGFBP3 ratio (41-155% higher risk). Conclusions: The 21-gene RS was related to favorable metabolic profiles including lower BMI, HOMA-IR, insulin, and C-peptide, and higher HDL in Chinese breast cancer patients, and its prognostic impact on breast cancer recurrence was more likely to present among patients with relatively favorable metabolic profiles.

Identifying Molecular Changes in Early Cervical Cancer Samples of Patients That Developed Metastasis.

Cervical cancer is one of the most common cancers in women worldwide. Patients diagnosed with early-stage cervical cancer have a good prognosis, however, 10-20% suffer from local or distant recurrent disease after primary treatment. Treatment options for recurrent cervical cancer are limited. Therefore, it is crucial to identify factors that can predict patients with an increased risk of recurrence to optimize treatment to prevent the recurrence of cervical cancer. We aimed to identify biomarkers in early-stage primary cervical cancer which recurred after surgery. Formalin-Fixed, Paraffin-Embedded surgical specimens of 34 patients with early-stage cervical cancer (FIGO 2009 stage 1B1) and 7 healthy controls were analyzed. Targeted gene expression profiling using the PanCancer IO 360 panel of NanoString Technology was performed. The findings were confirmed by performing immunohistochemistry stainings. Various genes, namely GLS, CD36, WNT5a, HRAS, DDB2, PIK3R2, and CDH2 were found to be differentially highly expressed in primary cervical cancer samples of patients who developed distant recurrence. In addition, The relative infiltration score of CD8+ T cells, CD80+CD86+ macrophages, CD163+MRC1+ macrophages, and FOXP3+IL2RA+ regulatory T cells were significantly higher in this group of samples. In contrast, no significant differences in gene expression and relative immune infiltration were found in samples of patients who developed local recurrence. The infiltration of CD8 and FOXP3 cells were validated by immunohistochemistry using all samples included in the study. We identified molecular alterations in primary cervical cancer samples from patients who developed recurrent disease. These findings can be utilized towards developing a molecular signature for the early detection of patients with a high risk to develop metastasis.

Large-Scale Gastric Cancer Susceptibility Gene Identification Based on Gradient Boosting Decision Tree.

The early clinical symptoms of gastric cancer are not obvious, and metastasis may have occurred at the time of treatment. Poor prognosis is one of the important reasons for the high mortality of gastric cancer. Therefore, the identification of gastric cancer-related genes can be used as relevant markers for diagnosis and treatment to improve diagnosis precision and guide personalized treatment. In order to further reveal the pathogenesis of gastric cancer at the gene level, we proposed a method based on Gradient Boosting Decision Tree (GBDT) to identify the susceptible genes of gastric cancer through gene interaction network. Based on the known genes related to gastric cancer, we collected more genes which can interact with them and constructed a gene interaction network. Random Walk was used to extract network association of each gene and we used GBDT to identify the gastric cancer-related genes. To verify the AUC and AUPR of our algorithm, we implemented 10-fold cross-validation. GBDT achieved AUC as 0.89 and AUPR as 0.81. We selected four other methods to compare with GBDT and found GBDT performed best.

The predictive prognostic values of CBFA2T3, STX3, DENR, EGLN1, FUT4, and PCDH7 in lung cancer.

BACKGROUND: Lung cancer is one of the most malignant tumors. However, neither the pathogenesis of lung cancer nor the prognosis markers are completely clear. The purpose of this study is to screen the diagnostic or prognostic markers of lung cancer. METHODS: TCGA and GEO datasets were used to analyze the relationship between lung cancer-related genes and lung cancer samples. Common differential genes were screened, and a univariate Cox regression analysis was used to screen survival related genes. A univariable Cox proportional hazards regression analysis was used to verify the genes and construct risk model. The key factors affecting the prognosis of lung cancer were determined by univariate and multivariate regression analyses. The ROC curve, AUC and the survival of each risk gene was analyzed. Finally, the biological functions of high- and low-risk patients were explored by GSEA and an immune-infiltration analysis. RESULTS: Based on the common differential genes, 13 genes significantly related to lung cancer survival were identified. Eight risk genes (CBFA2T3, DENR, EGLN1, FUT2, FUT4, PCDH7, PHF14, and STX3) were screened out. The results showed that risk status may be an independent prognostic factor, and the risk score predicted the prognosis of lung cancer. CBFA2T3 and STX3 are protective genes, while DENR, EGLN1, FUT4 and PCDH7 are dangerous genes. These 6 genes can be used as independent lung cancer prognosis markers. The corresponding biological functions of genes expressed in high-risk patients were mostly related to tumor proliferation and inflammatory infiltration. Neutrophil, CD8+T, Macrophage M0, Macrophage M1- and mDC-activated cells were high in high-risk status samples. CONCLUSIONS: CBFA2T3, STX3, DENR, EGLN1, FUT4, and PCDH7 are important participants in the occurrence and development of lung cancer. High-risk patients display serious inflammatory infiltration. This study not only provides insight into the mechanism of occurrence and development of lung cancer, but also provides potential targets for targeted therapy of lung cancer.

Expanded analysis of secondary germline findings from matched tumor/normal sequencing identifies additional clinically significant mutations.

BACKGROUND: Next-generation sequencing (NGS) for tumor molecular profiling can reveal secondary germline pathogenic and likely pathogenic variants (LPV/PV). The American College of Medical Genetics (ACMG) recommends return of secondary results for a subset of 59 genes, but other genes with evidence of clinical utility are emerging. We previously reported that 4.3% of patients who underwent NGS of a targeted panel of 201 genes had LPV/PV based on the ACMG list. Here we report the frequency of additional germline cancer-related gene variants and discuss their clinical utility. PATIENTS AND METHODS: Matched tumor and germline DNA NGS of a targeted panel of 201 genes was performed in a research laboratory on samples from 1000 patients with advanced or metastatic solid tumors enrolled in a molecular testing protocol (NCT01772771). The frequency of germline LPV/PV in 54 cancer-related genes, beyond the genes in ACMG list, were analyzed. RESULTS: Among 1000 patients who underwent tumor/normal DNA sequencing, 46 (4.6%) were found to have a germline LPV/PV in the following genes: AR-(5), ATM-(4), BAP1-(1), CDH1-(1), CDKN2A-(1), CHEK1-(2), CHEK2-(10), EGFR-(1), ERCC3-(4), ERCC5-(1), HNF1B-(1), HRAS-(1), MITF-(4), MLL3-(1), NF1-(3), PKHD1-(4), PTCH1-(1), and SMARCA4-(1). Thus, a total 8.7% of patients had an LPV/PV with 2 patients having 2 concomitant germline LPV/PV. Five mutations in high-penetrance hereditary cancer predisposition genes were selected to be returned to patients or their representatives: BAP1, CDH1, CDKN2A, EGFR, and SMARCA4. CONCLUSIONS: Broader genomic testing is likely to identify additional secondary pathogenic germline alterations, some with potential clinical utility for return to patients and their relatives. The recommended genes for which germline results should be returned are continually changing, warranting continued study.

A High-throughput Quantitative Expression Analysis of Cancer-related Genes in Human HepG2 Cells in Response to Limonene, a Potential Anticancer Agent.

BACKGROUND: Citrus bioactive compounds, as active anticancer agents, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. OBJECTIVES: The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. METHODS: The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of the pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. Highthroughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. RESULTS: In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene- driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from the most to the least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. CONCLUSION: The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines.

Detection of oncogenic mutations in resected bronchial margins by next-generation sequencing indicates early relapse in stage IA lung adenocarcinoma patients.

Stage I non-small cell lung cancer (NSCLC) patients experience a relatively high rate of recurrence, ranging from about 30-35%. We hypothesized that this elevated risk of recurrence is due to the presence of tumor cells at bronchial margins which was undetected by conventional light microscopy.Patients with clinical stage IA (T1N0M0) NSCLC were enrolled in this study,which included 8 early-relapse(ER) and 6 no-relapse(NR) patients. Primary tumor, bronchial margin,and normal lung tissues were collected and sent to a central site for targeted next-generation sequencing analysis. All of the patients were lung adenocarcinoma. Gene mutations were identified in all tumor tissue samples (100%).Oncogenic mutations were identified in 87.5%(7/8) bronchial margins of early relapse patients,whereas only 16.7%(1/6) no-relapse (NR) patient of marginal tissue had identified gene mutation.Additionally, concordance between primary tumor and bronchial margins was relatively high, with 4 of 8 (50%) ER patients having at least one identical mutation. Moreover, according to the gene mutation status in marginal tissue, 87.5% (7/8) of patients with at least one gene mutation in the bronchial margins had local recurrence or metastasis,whereas only 16.7% (1/6) of patients without any mutation detected had signs of relapse,the recurrence rate was significantly higher than that of the negative mutation margin group ((p (log-rank) = 0.023). The existence of oncogenic mutations in bronchial margins may represent occult residual tumor and elevated risk of recurrence in early stage NSCLC patients.Thus,assessing molecular status in bronchial margins may help identify patients who might benefit from extensive surgery or adjuvant treatment.

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.