Identification and clinical significance of somatic oncogenic mutations in epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (EOC) is a heterogeneous disease with diverse clinicopathological features and behaviors, and its heterogeneity may be concerned with the accumulation of multiple somatic oncogenic mutations. The major goals of this study are to systematically perform the comprehensive mutational profiling in EOC patients, and investigate the associations between somatic mutations and clinicopathological characteristics. METHODS: A total of 80 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery, and genomic DNAs were extracted from fresh-frozen tissues. We investigated mutational status in hot spot regions of 50 cancer-related genes by targeted next-generation sequencing using an Ion AmpliSeq Cancer Hotspot Panel v2 Kit. RESULTS: Validated mutations were detected in 66 of the 80 tumors (82.5%). The five most frequently mutated genes were TP53 (43.8%), PIK3CA (27.5%), KRAS (23.8%), PTEN (10%) and CTNNB1 (10%). PTEN and CTNNB1 mutations were associated with younger age. PIK3CA1, KRAS and CTNNB1 mutations were observed in early-stage, whereas TP53 mutations were more common in advanced stage. Significant associations were observed between TP53 mutation and serous carcinoma, and between KRAS mutation and mucinous carcinoma. Both PIK3CA mutation and CTNNB1 mutation were also significantly associated with endometrioid and clear cell carcinoma. The patients with PIK3CA and KRAS mutations were significantly associated with favorable progression free survival (PFS). In particular, PIK3CA mutations had more significant associations with favorable PFS than PIK3CA wild-type in the endometrioid subtype (P = 0.012). Patients with mutations only in TP53 were significantly associated with worse PFS. CONCLUSION: EOCs were heterogeneous at the genomic level and harbored somatic oncogenic mutations. Our molecular profiling may have the potential for becoming a novel stratification within histological subtypes of EOC. Further studies are needed to define molecular classification for improved clinical outcomes and treatment of EOC patients in future.

Tumor ß-catenin expression is associated with immune evasion in non-small cell lung cancer with high tumor mutation burden.

ß-catenin expression by tumor cells suppressed dendritic cell recruitment to the tumor microenvironment in a melanoma model, resulting in fewer tumor-infiltrating lymphocytes. Immunohistochemistry was used in the present study to examine the association between the expression of ß-catenin and tumor infiltrating lymphocytes and CD11c+ cells in 122 patients with non-small cell lung cancer (NSCLC), who underwent radical surgery. ß-catenin was positive in 24% of NSCLC tumors compared with 59% of squamous cell carcinomas and 11% of adenocarcinomas. There was no significant association between the expression of ß-catenin and the frequency of CD8+ cell infiltration into tumor tissues, including the stroma. Conversely, the infiltration of CD8+ cells into tumor nests was significantly lower in ß-catenin-positive cases compared with that in negative ß-catenin cases. Similarly, CD11c+ cell infiltration was significantly lower in the ß-catenin-positive group. The ß-catenin-positive group had shorter overall survival and recurrence-free survival times compared with that in the negative group. Furthermore, ß-catenin-positive NSCLC had a high tumor mutation burden, but tended to have a low expression of programmed death-ligand 1. In conclusion, the expression of ß-catenin in NSCLC was negatively associated with CD11c+ cells and cytotoxic T cell infiltration at the tumor site and had a tendency towards a poor prognosis.

Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma.

PURPOSE: Endometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients. METHODS: A total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes. RESULTS: Validated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion. CONCLUSION: Our comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.

Clinical implication of oncogenic somatic mutations in early-stage cervical cancer with radical hysterectomy.

It is well known that tumour initiation and progression are primarily an accumulation of genetic mutations. The mutation status of a tumour may predict prognosis and enable better selection of targeted therapies. In the current study, we analysed a total of 55 surgical tumours from stage IB-IIB cervical cancer (CC) patients who had undergone radical hysterectomy including pelvic lymphadenectomy, using a cancer panel covering 50 highly mutated tumorigenesis-related genes. In 35 patients (63.6%), a total 52 mutations were detected (58.3% in squamous cell carcinoma, 73.7% in adenocarcinoma), mostly in PIK3CA (34.5%) and KRAS and TP53 (9.1%). Being mutation-positive was significantly correlated with pelvic lymph node (PLN) metastasis (P = 0.035) and tended to have a worse overall survival (P = 0.076). In particular, in the patients with squamous cell carcinoma, there was a significant association between being mutation-positive and relapse-free survival (P = 0.041). The patients with PLN metastasis had a significantly worse overall survival than those without (P = 0.006). These results indicate that somatic mutation status is a predictive biomarker for PLN metastasis in early-stage CC, and is consequently related to poor prognosis. Therefore, comprehensive genetic mutations, rather than a single genetic mutation, should be examined widely in order to identify novel genetic indicators with clinical usefulness.

Tumor mutation burden and immunological, genomic, and clinicopathological factors as biomarkers for checkpoint inhibitor treatment of patients with non-small-cell lung cancer.

Cancer treatment using immune checkpoint inhibitors is widely used, although biomarkers predictive of response are not well established. However, both the expressions of programmed cell death ligand 1 (PD-L1) and the tumor mutation burden (TMB) hold promise as such biomarkers for immune checkpoint inhibitors; however, its characteristics and clinical and immunological impacts have not been fully analyzed. We, therefore, evaluated the clinical and immunological parameters related to TMB to identify potential new biomarkers. We enrolled 92 patients with non-small-cell lung cancer who underwent surgery at Fukushima Medical University Hospital from 2013 to 2016. TMB of individual tumors was calculated by whole-exome sequencing analysis. Major cancer-related gene mutations were evaluated using panel sequencing. Expression of PD-L1 and abundance of tumor-infiltrating lymphocytes were evaluated by immunohistochemistry using surgical samples. The median TMB value was 60. TMB was significantly higher in men, current or former smokers, and in patients with squamous cell carcinoma, tumor size ≥ 2.8 cm, wild-type EGFR, TP53 gene mutation-positive status, and cyclin-dependent kinase-inhibitor gene 2A mutation-positive status. According to multivariate analysis, TMB was significantly associated with EGFR gene mutation-negative status (p = 0.0111) and TP53 gene mutation-positive status (p = 0.0425). If TMB is identified as a robust biomarker for immune checkpoint inhibitor administration, analysis of TP53 and EGFR mutations may provide a relatively rapid and easy proxy for predicting TMB.

Prognostic Impact of Tumor Mutation Burden in Patients With Completely Resected Non-Small Cell Lung Cancer: Brief Report.

INTRODUCTION: Tumor mutation burden (TMB) is thought to be associated with the amount of neoantigen in the tumor and to have an important role in predicting the effect of immune checkpoint inhibitors. However, the relevance of TMB to prognosis is not yet fully understood. In this study, we investigated the clinical significance of TMB in patients with NSCLC and examined the relationship between TMB and prognosis. METHODS: We calculated TMB within individual tumors by whole-exome sequencing analysis using next-generation sequencing. We included that there were 90 patients with NSCLC who underwent surgery in the Hospital of Fukushima Medical University from 2013 to 2016. No patients received chemotherapy or immunotherapy before surgery. We assessed the correlation between TMB and prognosis. RESULTS: TMB greater than 62 was associated with worse overall survival (OS) of patients with NSCLC (hazard ratio [HR] = 6.633, p = 0.0003). Multivariate analysis showed poor prognosis with high TMB (HR = 12.31, p = 0.019). In patients with stage I NSCLC, higher TMB was associated with worse prognosis for both OS (HR = 7.582, p = 0.0018) and disease-free survival (HR = 6.07, p = 0.0072). CONCLUSIONS: High TMB in NSCLC is a poor prognostic factor. If high TMB is a predictor of the efficacy of immune checkpoint inhibitors, postoperative adjuvant therapy with immune checkpoint inhibitors may contribute to improvement of recurrence and OS.

[Exploring the Possibility of New Biomarkers of Immune Checkpoint Inhibitors for Non-Small Cell Lung Cancer (NSCLC)].

Mutation burden in a tumor, presumably involving neo-antigens in the tumor tissue, is also thought to be one of the better predictors for the efficacy of immune checkpoint inhibitors. However, it is difficult to analyze the mutation burden routinely in the clinic. Here, we describe more convenient factors that can be used as surrogate markers of mutation burden. Ninety-four patients with NSCLC who underwent resection in our institution were recruited for this study. Mutation burden and major gene alterations were analyzed by using next generation sequencing. Several immunological parameters were also assessed using immunohistochemistry. Statistical analysis was performed on mutation burden, major gene alternations, immunohistochemistry, and clinical parameters. The median mutation load was 54 mutations(range, 10-363 mutations). Squamous cell carcinoma, EGFRmutation -negativity, and TP53 alteration-positivity were closely connected with higher mutation burden. Multiple regression analysis showed that mutation burden in the tumor could be associated with EGFRmutation and TP53 alteration status.

Identification of a long non-coding RNA as a novel biomarker and potential therapeutic target for metastatic prostate cancer.

Metastatic prostate cancer (PCa) is still an incurable disease. Long non-coding RNAs (lncRNAs) may be an overlooked source of cancer biomarkers and therapeutic targets. We therefore performed RNA sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The most highly up-regulated transcript was LOC728606, a lncRNA now designated PCAT18. PCAT18 is specifically expressed in the prostate compared to 11 other normal tissues (p<0.05) and up-regulated in PCa compared to 15 other neoplasms (p<0.001). Cancer-specific up-regulation of PCAT18 was confirmed on an independent dataset of PCa and benign prostatic hyperplasia samples (p<0.001). PCAT18 was detectable in plasma samples and increased incrementally from healthy individuals to those with localized and metastatic PCa (p<0.01). We identified a PCAT18-associated expression signature (PES), which is highly PCa-specific and activated in metastatic vs. primary PCa samples (p<1E-4, odds ratio>2). The PES was significantly associated with androgen receptor (AR) signalling. Accordingly, AR activation dramatically up-regulated PCAT18 expression in vitro and in vivo. PCAT18 silencing significantly (p<0.001) inhibited PCa cell proliferation and triggered caspase 3/7 activation, with no effect on non-neoplastic cells. PCAT18 silencing also inhibited PCa cell migration (p<0.01) and invasion (p<0.01). These results position PCAT18 as a potential therapeutic target and biomarker for metastatic PCa.

Alternative splicing of genes during neuronal differentiation of NT2 pluripotential human embryonal carcinoma cells.

We analyzed the mRNA diversity of genes after inducing neuronal differentiation in human NT2 teratocarcinoma cells using all-trans retinoic acid (RA). DNA microarray analyses of cells treated with RA identified 358 RA-responsive genes. mRNA diversity analysis revealed that 274 genes produced multiple protein-coding transcripts by alternative splicing. Among these 274 genes, we chose 26 genes that showed AS in their C-terminus and 12 transcription factor genes for further analysis. By using transcript-specific primers, we performed quantitative real-time PCR analysis to examine the expression profiles of all the protein-coding transcripts. Consequently, we identified genes which showed different RA-induced changes in the expression of their protein-coding transcripts.

Identification and functional analyses of 11,769 full-length human cDNAs focused on alternative splicing.

We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5'-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment.

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

A novel estrogen receptor-related protein gamma splice variant lacking a DNA binding domain exon modulates transcriptional activity of a moderate range of nuclear receptors.

A novel estrogen receptor-related protein (ERR) gamma splice variant cDNA (ERRgamma3) was found in human full-length cDNA libraries. ERRgamma3 cDNA consists of 3362 base pairs and has an open reading frame of 1188bp. The predicted peptide sequence of ERRgamma3 differs from both ERRgamma1 and ERRgamma2 in missing 39 amino acid residues corresponding to the second zinc finger motif of the DNA binding domain (DBD). ERRgamma3 gene consists of 8 exons including three unique 5'-terminal exons and lacks the exon encoding the second zinc finger motif. The expression of ERRgamma3 was confined to adipocytes and prostate while that of ERRgamma2 was fairly widespread. The ERRgamma3 product was shown by transactivation assay to have no ability to activate ERE-controlled transcription. However, ERRgamma3 has an ability to modulate the transcriptional activity of other nuclear hormone receptors. ERRgamma3 augmented the ligand-dependent transcriptional activities of ER (estrogen receptor) alpha, ERbeta, and thyroid receptor (TR) alpha by 1.3-, 4-, and 2.1-fold whereas it inhibited fully the activity of glucocorticoid receptor (GR). However, ERRgamma3 had no effect on Vitamin D3 receptor, retinoic acid receptor alpha or peroxisome proliferator activated receptor alpha, beta, and gamma. These findings will help to elucidate the physiological role of the ERRgamma subfamily.

Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities.

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-alpha and salusin-beta. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-alpha or salusin-beta to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-beta stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-alpha and salusin-beta are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis.

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.

cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFalpha.

Gene expression of synoviocytes stimulated with tumor necrosis factor-alpha (TNFalpha) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFalpha stimulation. These findings suggest that the synoviocyte response to TNFalpha stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.