Rapid detection of epidermal growth factor receptor mutations with multiplex PCR and primer extension in lung cancer

Epidermal growth factor receptor (EGFR) kinase domain mutations hyperactivate the kinase and confer kinase addiction of the non-small-cell lung cancer (NSCLC) tumor cells. Almost all of these mutations are located within exons 18-21. The -216 single nucleotide polymorphism in the promoter region is associated with increased EGFR production. We present a method for detecting these common mutations in 81 cases of NSCLC. The protocol is based on the multiplex amplification of promoter region and exons 18-21 of the EGFR genes in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at -216 promoter region and codons 719, 746-750, 790, 858 of the EGFR gene. We compared the results with that from direct sequencing for detecting EGFR mutations in 81 cases of NSCLC. The two methods identified the same 26 mutations, but our method is superior to direct sequencing in terms of the amount of work and time required. We presented a simple and fast method to detect mutations of EGFR genes in NSCLC

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Using primer extension method to detect the mutations of RAS signaling pathway-related genes and clinical application

RAS/RAF/MEK/ERK訊息傳遞路徑在各種細胞中調節細胞週期的行進和細胞凋亡。在轉型細胞株和人類腫瘤中這條訊息傳遞路徑經常發生突變。人類腫瘤經常會表現RAS蛋白質，RAS蛋白質經常借由點突變而活化。約有20%的癌症至少會有一種RAS基因發生突變。突變通常發生在胺基酸密碼組第12、13和61的位置。活化的RAS會與RAF結合，開啟RAF的活化作用，BRAF最常發生突變的位置是在胺基酸密碼組第600位置。在大腸直腸癌中，KRAS基因突變率是最高的，接著是NRAS，到目前為止還沒有發現HRAS基因的突變。HRAS是3種RAS基因中唯一和口腔癌有關聯的。一開始，我們先建立偵測K-、N- 和H-RAS突變熱點的方法。結果我們證實：我們的方法提供臨床上可以用來評估K-、N- 和H-RAS是否突變的一個簡單、快速且可信賴的方法。接著，我們延伸了這個方法的應用，在一個反應管中，同時放大3種RAS基因的第二外顯子胺基酸密碼組第12和13位置與第三外顯子胺基酸密碼組第61位置和BRAF基因第十五外顯子胺基酸密碼組第600位置。多重聚合酶連鎖反應後將產物純化，接著將純化後產物分成三個部分。每個部分利用七條和二條不同長度的RAS引子和BRAF引子，分別測三種RAS基因的胺基酸密碼組第12、13和61位置和BRAF基因的胺基酸密碼組第600位置是否突變。我們將引子延伸法和直接定序的方法拿來互相比較。結果顯示這兩個方法的結果是一致的，不過以所需工作量和時間而論，我們的方法比直接定序的方法好。依據這個方法的原理，將來可以用來偵測其他基因在大腸直腸癌、口腔癌或其他各種不同癌症是否突變。The RAS/RAF/MEK/ERK signal transduction pathway regulates cell cycle progression and apoptosis in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and frequently linked with human cancers. Human tumors very frequently express Ras proteins that have been activated by point mutation. It is estimated that ~20% of all tumors have undergone an activating mutation in one of the three RAS genes. Almost all of these mutations are located at codons 12, 13 and 61. Three RAF genes are regulated by binding RAS and the most common mutation is at codon 600 of BRAF gene. In colorectal cancer, KRAS gene is the predominantly mutated RAS gene followed by NRAS, whereas HRAS mutation has never been identified. HRAS is the only RAS gene that has been implicated in oral cancer. First, we present a method for detecting K-, N- and H-RAS hotspot mutations. The method that we demonstrated in this report provides a simple, fast and reliable way to identify K-, N- and H-RAS mutations. We extended the application using multiplex amplification of codons 12 and 13 of exon 2, codon 61 of exon 3 of three RAS genes and codon 600 of exon 15 of BRAF gene in a single tube. The products were cleaned and then split in three tubes. Each tube was subjected for primer extension using seven and two different-sized RAS and two BRAF primers respectively for different RAS gene and BRAF gene separately to detect base changes in codons 12, 13 and 61 of each RAS gene and codon 600 of BRAF gene. We compared the results with that from direct sequencing for detecting K-, N-, H-RAS and BRAF mutations. Threse two methods yield identical results, but our method is superior to direct sequencing in terms of the amount of work and time required. Meanwhile, the principle of the method can also be used to detect the mutation at a given codon in other genes in colorectal, oral and other cancers.中文摘要 i 英文摘要 ii 第一章 序論 1 第一節 癌症與癌症之生成 1 第二節 大腸癌的簡介 7 一. 大腸癌的流行病學特徵 7 二. 大腸癌的病因 10 三. 大腸癌的症狀 12 四. 大腸癌的診斷 13 五. 大腸癌的治療 15 六. 大腸癌形成的分子機轉 19 第三節 RAS訊息傳遞 24 第四節 研究目的 28 第二章 研究材料與方法 30 第一節 病人及檢體收集 30 第二節 DNA萃取 30 第三節 直接定序 31 第四節 引子延伸法 33 一. 利用多重PCR (multiplex PCR) 及多重引子延伸法 (mutiplex primer extension) 來分別偵測不同RAS的hotspot mutations 33 二. 利用RAS通用引子 (universal primer) 多重PCR同時放大K-、N-和H-RAS Exon 2和Exon 3及多重引子延伸法來分別偵測不同RAS的hotspot mutations 39 三. 利用RAS通用引子和BRAF引子多重PCR及多重引子延伸法來分別偵測不同RAS及BRAF的hotspot mutations 42 第五節 統計方式 44 第三章 研究結果 45 第一節. 利用直接定序的方法分析RAS及BRAF的基因突變 45 一. KRAS基因 45 二. NRAS基因 46 三. HRAS基因 47 四. BRAF基因 47 第二節. 利用多重引子延伸法分析RAS及BRAF基因的突變 47 一. 利用多重PCR及多重引子延伸法來分別偵測不同RAS的hotspot mutations 47 二. 利用RAS通用引子多重PCR同時放大K-、N-和H-RAS Exon 2和Exon 3及多重引子延伸法來分別偵測不同RAS的hotspot mutations 50 三. 利用RAS通用引子和BRAF引子多重PCR及多重引子延伸法來分別偵測不同RAS及BRAF的hotspot mutations 52 第三節. 靈敏度測試 53 第四節. 統計結果 54 第四章 討論 56 參考書目 9

Male-Specific Long Noncoding RNA TTTY15 Inhibits Non-Small Cell Lung Cancer Proliferation and Metastasis via TBX4

Gender affects cancer susceptibility. Currently, there are only a few studies on Y chromosome-linked long noncoding RNAs (lncRNAs), and the potential association between lncRNAs and cancers in males has not been fully elucidated. Here, we examined the expression of testis-specific transcript Y-linked 15 (TTTY15) in 37 males with non-small cell lung cancer (NSCLC), and performed circular chromosome conformation capture with next-generation sequencing to determine the genomic interaction regions of the TTTY15 gene. Our results showed that the expression levels of TTTY15 were lower in NSCLC tissues. Lower TTTY15 expression levels were associated with Tumor-Node-Metastasis (TNM) stage. A TTTY15 knockdown promoted malignant transformation of NSCLC cells. Based on the bioinformatics analysis of circular chromosome conformation capture data, we found that T-box transcription factor 4 (TBX4) may be a potential target gene of TTTY15. The RNA immunoprecipitation and chromatin immunoprecipitation results showed that TTTY15 may interact with DNA (cytosine-5)-methyltransferase 3A (DNMT3A), and the TTTY15 knockdown increased the binding of DNMT3A to the TBX4 promoter. We concluded that low TTTY15 expression correlates with worse prognosis among patients with NSCLC. TTTY15 promotes TBX4 expression via DNMT3A-mediated regulation. The identification of lncRNAs encoded by male-specific genes may help to identify potential targets for NSCLC therapy

Molecular Classification of Hepatocellular Carcinoma Using Wnt–Hippo Signaling Pathway-Related Genes

In Taiwan, a combination of hepatitis B and C infection, economic boom-related food and alcohol overconsumption, and Chinese medicine prescriptions has led to a high rate of hepatocellular carcinoma (HCC). However, the causative factors and underlying tumor biology for this unique HCC environment have not been identified. Wnt and Hippo signaling pathways play an important regulatory role in HCC development, and their functions are generally considered as positive and negative regulators of cell proliferation, respectively. In this study, we characterized the molecular features of HCC using a newly developed classification system based on the expression of the Wnt–Hippo signaling pathway-related genes. RNA sequencing (RNA-Seq) was performed on liver tumor tissues from 100 patients with liver cancer. RNA-Seq data for 272 previously characterized Wnt–Hippo signaling pathway-related genes were used for hierarchical clustering. We analyzed the data in terms of prognostic value, transcriptome features, immune infiltration, and clinical characteristics, and compared the resulting subclasses with previously published classifications. Four subclasses of HCC (HCCW1–4) were identified. Subclass HCCW1 displayed the highest PCDHB4 expression. Subclass HCCW2 displayed lower Edmondson–Steiner grades (I and II) and CTNNB1 mutation frequencies. Subclass HCCW3 was associated with a good prognosis, the highest PCDHGB7 expression, high CD8+ naïve T cells abundance, and relatively low TP53 mutation rates. Subclass HCCW4 was associated with a poor prognosis, the highest PCDHB2 and PCDHB6 expression, a relatively high abundance of Th1 cells, NKT and class-switched memory B cells, relatively low enrichment of cDC, iDC, and CD4+ memory T cells, and high Edmondson–Steiner grades (III and IV). We also identified Wnt–Hippo signaling pathway-related genes that may influence immune cell infiltration. We developed a panel of 272 Wnt–Hippo signaling pathway-related genes to classify HCC into four groups based on Taiwanese HCC and The Cancer Genome Atlas Liver Hepatocellular Carcinoma datasets. This novel molecular classification system may aid the treatment of HCC

Metatranscriptomic Analysis of Human Lung Metagenomes from Patients with Lung Cancer

This study was designed to characterize the microbiomes of the lung tissues of lung cancer patients. RNA-sequencing was performed on lung tumor samples from 49 patients with lung cancer. Metatranscriptomics data were analyzed using SAMSA2 and Kraken2 software. 16S rRNA sequencing was also performed. The heterogeneous cellular landscape and immune repertoires of the lung samples were examined using xCell and TRUST4, respectively. We found that nine bacteria were significantly enriched in the lung tissues of cancer patients, and associated with reduced overall survival (OS). We also found that subjects with mutations in the epidermal growth factor receptor gene were less likely to experience the presence of Pseudomonas. aeruginosa. We found that the presence of CD8+ T-cells, CD4+ naive T-cells, dendritic cells, and CD4+ central memory T cells were associated with a good prognosis, while the presence of pro B-cells was associated with a poor prognosis. Furthermore, high clone numbers were associated with a high ImmuneScore for all immune receptor repertoires. Clone numbers and diversity were significantly higher in unpresented subjects compared to presented subjects. Our results provide insight into the microbiota of human lung cancer, and how its composition is linked to the tumor immune microenvironment, immune receptor repertoires, and OS