Multiomics identifies the link between intratumor steatosis and the exhausted tumor immune microenvironment in hepatocellular carcinoma.

BACKGROUND AND AIMS: Immunotherapy has become the standard-of-care treatment for hepatocellular carcinoma (HCC), but its efficacy remains limited. To identify immunotherapy-susceptible HCC, we profiled the molecular abnormalities and tumor immune microenvironment (TIME) of rapidly increasing nonviral HCC. APPROACHES AND RESULTS: We performed RNA-seq of tumor tissues in 113 patients with nonviral HCC and cancer genome sequencing of 69 genes with recurrent genetic alterations reported in HCC. Unsupervised hierarchical clustering classified nonviral HCCs into three molecular classes (Class I, II, III), which stratified patient prognosis. Class I, with the poorest prognosis, was associated with TP53 mutations, whereas class III, with the best prognosis, was associated with cadherin-associated protein beta 1 (CTNNB1) mutations. Thirty-eight percent of nonviral HCC was defined as an immune class characterized by a high frequency of intratumoral steatosis and a low frequency of CTNNB1 mutations. Steatotic HCC, which accounts for 23% of nonviral HCC cases, presented an immune-enriched but immune-exhausted TIME characterized by T cell exhaustion, M2 macrophage and cancer-associated fibroblast (CAF) infiltration, high PD-L1 expression, and TGF-ß signaling activation. Spatial transcriptome analysis suggested that M2 macrophages and CAFs may be in close proximity to exhausted CD8+ T cells in steatotic HCC. An in vitro study showed that palmitic acid-induced lipid accumulation in HCC cells upregulated PD-L1 expression and promoted immunosuppressive phenotypes of cocultured macrophages and fibroblasts. Patients with steatotic HCC, confirmed by chemical-shift MR imaging, had significantly longer PFS with combined immunotherapy using anti-PD-L1 and anti-VEGF antibodies. CONCLUSIONS: Multiomics stratified nonviral HCCs according to prognosis or TIME. We identified the link between intratumoral steatosis and immune-exhausted immunotherapy-susceptible TIME.

MYEOV overexpression induced by demethylation of its promoter contributes to pancreatic cancer progression via activation of the folate cycle/c-Myc/mTORC1 pathway.

BACKGROUND: While molecular targeted drugs and other therapies are being developed for many tumors, pancreatic cancer is still considered to be the malignant tumor with the worst prognosis. We started this study to identify prognostic genes and therapeutic targets of pancreatic cancer. METHODS: To comprehensively identify prognostic genes in pancreatic cancer, we investigated the correlation between gene expression and cancer-specific prognosis using transcriptome and clinical information datasets from The Cancer Genome Atlas (TCGA). In addition, we examined the effects of the suppression of candidate prognostic genes in pancreatic cancer cell lines. RESULT: We found that patients with high expression levels of MYEOV, a primate-specific gene with unknown function, had significantly shorter disease-specific survival times than those with low expression levels. Cox proportional hazards analysis revealed that high expression of MYEOV was significantly associated with poor survival and was an independent prognostic factor for disease-specific survival in pancreatic cancer patients. Analysis of multiple cancer samples revealed that the MYEOV promoter region is methylated in noncancer tissues but is demethylated in tumors, causing MYEOV overexpression in tumors. Notably, the knockdown of MYEOV suppressed the expression of MTHFD2 and other folate metabolism-related enzyme genes required for the synthesis of amino acids and nucleic acids and also restored the expression of c-Myc and mTORC1 repressors. CONCLUSION: There is a significant correlation between elevated MYEOV expression and poor disease-specific survival in pancreatic cancer patients. MYEOV enhances the activation of several oncogenic pathways, resulting in the induction of pancreatic cancer cell proliferation. Overall, MYEOV acts as an oncogene in pancreatic cancer. Furthermore, MYEOV may be a prognostic biomarker and serve as an 'actionable' therapeutic target for pancreatic cancers.

Inhibition of EGFR and MEK surmounts entrectinib resistance in a brain metastasis model of NTRK1-rearranged tumor cells.

Tropomyosin receptor kinase (TRK) inhibitors have demonstrated histology-agnostic efficacy in patients with neurotrophic receptor tyrosine kinase (NTRK) gene fusion. Although responses to TRK inhibitors can be dramatic and durable, duration of response may eventually be limited by acquired resistance via several mechanisms, including resistance mutations such as NTRK1-G595R. Repotrectinib is a second-generation TRK inhibitor, which is active against NTRK1-G595R. However, its efficacy against entrectinib-resistant tumors has not been fully elucidated. In the present study, we established entrectinib-resistant tumor cells (M3B) in a brain metastasis model inoculated with NTRK1-rearranged KM12SM cells and examined the sensitivity of M3B cells to repotrectinib. While M3B cells harbored the NTRK1-G595R mutation, they were unexpectedly resistant to repotrectinib. The resistance was due to extracellular signal-regulated kinase (ERK) reactivation partially mediated by epidermal growth factor receptor (EGFR) activation. We further demonstrate that the triplet combination of repotrectinib, EGFR inhibitor, and MEK inhibitor could sensitize M3B cells in vitro as well as in a brain metastasis model. These results indicate that resistant mutations, such as NTRK1-G595R, and alternative pathway activation, such as ERK activation, could simultaneously occur in entrectinib-resistant tumors, thereby causing resistance to second-generation inhibitor repotrectinib. These findings highlight the importance of intensive examinations to identify resistance mechanisms and application of the appropriate combination treatment to circumvent the resistance.

Early dynamics of circulating tumor DNA predict chemotherapy responses for patients with esophageal cancer.

We investigated whether early circulating tumor DNA (ctDNA) changes, measured using digital PCR (dPCR), can predict later chemotherapy responses in esophageal squamous cell cancer (ESCC). We compared the dynamics of ctDNA and tumor volumes during chemotherapy in 42 ESCC. The accuracy of predictions of later chemotherapy responses was evaluated by the ratio of the variant allele frequency of ctDNA (post-/pre-ctDNA) and the total tumor volume (post-/pre-volume) before and after an initial chemotherapy cycle using a receiver-operating characteristic curve analysis. Total positive and negative objective responses (ORs) were defined as either >50 or ≤50% reductions, respectively, in the total tumor volume at the end of first-line chemotherapy. Mutation screening of 43 tumors from 42 patients revealed 96 mutations. The pretreatment dPCR-ctDNA data were informative in 38 patients, using 70 selected mutations (1-3 per patient). The areas under the curve (AUCs) for the post-/pre-volume and post-/pre-ctDNA levels used in predicting the total OR were 0.85 and 0.88, respectively. The optimal cutoff value of post-/pre-ctDNA was 0.13. In 20 patients with post-/pre-volume ≥50%, the total OR could be predicted by the post-/pre-ctDNA with high accuracy; the AUC by post-/pre-ctDNA was higher than that by post-/pre-volume (0.85 versus 0.76, respectively). Patients with low post-/pre-ctDNA (n = 18) had a significantly better overall survival rate than those with high post-/pre-ctDNA (n = 20; P = 0.03). Early ctDNA changes after an initial cycle of chemotherapy predict later responses to treatment with high accuracy in ESCC patients.

Claudin-6 is a single prognostic marker and functions as a tumor-promoting gene in a subgroup of intestinal type gastric cancer.

BACKGROUND: We aimed to identify novel tumor-promoting drivers highly expressed in gastric cancer (GC) that contribute to worsened prognosis in affected patients. METHODS: Genes whose expression was increased and correlated with worse prognosis in GC were screened using datasets from the Cancer Genome Atlas and Gene Expression Omnibus. We examined Claudin-6 (CLDN6) immunoreactivity in GC tissues and the effect of CLDN6 on cellular functions in GC cell lines. The mechanisms underlying GC-promoting function of CLDN6 were also investigated. RESULTS: CLDN6 was identified as a gene overexpressed in GC tumors as compared with adjacent non-tumorous tissues and whose increased expression was positively correlated with worse overall survival of GC patients, particularly those with Lauren's intestinal type GC, in data from multiple publicly available datasets. Additionally, membranous CLDN6 immunoreactivity detected in intestinal type GC tumors was correlated with worse overall survival. In CLDN6-expressing GC cells, silencing of CLDN6 inhibited cell proliferation and migration/invasion abilities, possibly via suppressing transcription of YAP1 and its downstream transcriptional targets at least in part. CONCLUSIONS: This study identified CLDN6 as a GC-promoting gene, suggesting that CLDN6 to be a possible single prognostic marker and promising therapeutic target for a subset of GC patients.

Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.

Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.

A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth.

BACKGROUND: Homeobox A5 (HOXA5), a member of the HOX family, plays an important role in tumor development and morphogenesis, although opposite effects on tumorigenesis have been observed, depending on the tissue type. In this study, we aimed to investigate the role of a novel transcript from the HOXA6-HOXA5 locus in colon cancer tumorigenesis. METHODS: Human colon cancer cell lines were analyzed using next generation sequencing-based targeted mRNA capture. The effects of overexpression and silencing of HOXA5 transcripts were evaluated in vitro and using a xenograft nude mouse model. RESULTS: We identified three novel transcripts (HOXA5 short, long 1, and long 2) transcribed from the HOXA6-HOXA5 locus in HCT116 colon cancer cells using next generation sequencing-based targeted mRNA capture. Knockdown of HOXA5 long 1 and long 2 transcripts did not affect cell growth, while selective silencing of HOXA5 short RNA inhibited cell growth independent of HOXA5 expression. Stable overexpression of HOXA5 short RNA promoted proliferation and migration of colon cancer cell lines HCT116, DLD1, and HT-29 and accelerated tumor growth in the xenograft mouse model. In vitro translation assays suggested HOXA5 short RNA was a functional long non-coding RNA (lncRNA). Consistent with these observations, expression of HOXA5 short RNA was upregulated in advanced colon cancer tissues. Ingenuity Pathway Analysis of differentially expressed genes between HOXA5 short RNA overexpressed and silenced HCT116 cells revealed that HOXA5 short RNA preferentially modified expression of epidermal growth factor (EGF) signal-related genes. Western blot analysis demonstrated that stable overexpression of HOXA5 short RNA increased EGF receptor levels and facilitated its phosphorylation in both HCT116 cells and xenograft tumors. CONCLUSIONS: Our results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.

MEG3 Long Noncoding RNA Contributes to the Epigenetic Regulation of Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines.

Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-ß (TGF-ß)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-ß-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-ß in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells.

A rare male patient with classic Rett syndrome caused by MeCP2_e1 mutation.

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically affecting females. It is mainly caused by loss-of-function mutations that affect the coding sequence of exon 3 or 4 of methyl-CpG-binding protein 2 (MECP2). Severe neonatal encephalopathy resulting in death before the age of 2 years is the most common phenotype observed in males affected by a pathogenic MECP2 variant. Mutations in MECP2 exon 1 affecting the MeCP2_e1 isoform are relatively rare causes of RTT in females, and only one case of a male patient with MECP2-related severe neonatal encephalopathy caused by a mutation in MECP2 exon 1 has been reported. This is the first reported case of a male with classic RTT caused by a 5-bp duplication in the open-reading frame of MECP2 exon 1 (NM_001110792.1:c.23_27dup) that introduced a premature stop codon [p.(Ser10Argfs*36)] in the MeCP2_e1 isoform, which has been reported in one female patient with classic RTT. Therefore, both males and females displaying at least some type of MeCP2_e1 mutation may exhibit the classic RTT phenotype.

Jmjd5 functions as a regulator of p53 signaling during mouse embryogenesis.

Genetic studies have shown that aberrant activation of p53 signaling leads to embryonic lethality. Maintenance of a fine balance of the p53 protein level is critical for normal development. Previously, we have reported that Jmjd5, a member of the Jumonji C (JmjC) family, regulates embryonic cell proliferation through the control of Cdkn1a expression. Since Cdkn1a is the representative p53-regulated gene, we have examined whether the expression of other p53 target genes is coincidentally upregulated with Cdkn1a in Jmjd5-deficient embryos. The expression of a subset of p53-regulated genes was increased in both Jmjd5 hypomorphic mouse embryonic fibroblasts (MEFs) and Jmjd5-deficient embryos at embryonic day 8.25 without the induced expression of Trp53. Intercrossing of Jmjd5-deficient mice with Trp53 knockout mice showed that the growth defect of Jmjd5 mutant cells was significantly recovered under a Trp53 null genetic background. Chromatin immunoprecipitation analysis in Jmjd5 hypomorphic MEFs indicated the increased recruitment of p53 at several p53 target gene loci, such as Cdkn1a, Pmaip1, and Mdm2. These results suggest that Jmjd5 is involved in the transcriptional regulation of a subset of p53-regulated genes, possibly through the control of p53 recruitment at the gene loci. In Jmjd5-deficient embryos, the enhanced recruitment of p53 might result in the abnormal activation of p53 signaling leading to embryonic lethality.

Examination of a plasmid-based reverse genetics system for human astrovirus.

A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5' ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection.

EED regulates epithelial-mesenchymal transition of cancer cells induced by TGF-ß.

Histone methylation is involved in various biological and pathological processes including cancer development. In this study, we found that EED, a component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in epithelial-mesenchymal transition (EMT) of cancer cells induced by Transforming Growth Factor-beta (TGF-ß). The expression of EED was increased during TGF-ß-induced EMT and knockdown of EED inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. EED knockdown antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB1, ZEB2 and microRNA-200 (miR-200) family. Chromatin immunoprecipitation assays showed that EED was implicated in TGF-ß-induced transcriptional repression of CDH1 and miR-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of EED, which regulates PRC2 activity and histone methylation during TGF-ß-induced EMT of cancer cells.

Long Noncoding RNA RP11-278A23.1, a Potential Modulator of p53 Tumor Suppression, Contributes to Colorectal Cancer Progression.

Recently, many studies revealed that long noncoding RNAs (lncRNAs) play important roles in cancers. To identify lncRNAs contributing to colorectal cancers, we screened lncRNAs through expression and survival analyses in datasets from The Cancer Genome Atlas (TCGA). The screen revealed that RP11-278A23.1 expression is significantly increased in colorectal cancer tissues compared with normal tissues and that high RP11-278A23.1 expression correlates with poor prognosis. The knockdown of RP11-278A23.1 inhibited the growth of and promoted apoptosis in colorectal cancer cells. Next, to comprehensively examine differentially expressed genes after RP11-278A23.1 knockdown, RNA sequencing was performed in HCT116 cells. The expression of p21, a p53 target gene, was significantly upregulated, and the expression of several p53 target proapoptotic genes was also altered. RP11-278A23.1 knockdown increased p53 expression at the translational level but not at the transcriptional level. Interestingly, RP11-278A23.1 knockdown also altered the expression of these proapoptotic genes in DLD1 cells with mutated p53 and in p53-knockout HCT116 cells. These results suggest that RP11-278A23.1 modifies the expression of these apoptosis-related genes in p53-dependent and p53-independent manners. In summary, lncRNA RP11-278A23.1 contributes to colorectal cancer progression by promoting cell growth and inhibiting apoptosis, suggesting that this lncRNA may be a useful therapeutic target.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR.

There are limited methods to stably analyze the interactions between cancer cells and glial cells in vitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

Roles of histone methyl-modifying enzymes in development and progression of cancer.

Retroviral insertional mutagenesis in mice is considered a powerful forward genetic strategy to identify disease genes involved in cancer. Our high-throughput screens led to frequent identification of the genes encoding the enzymes engaged in histone lysine methylation. Histone methylation can positively or negatively impact on gene transcription, and then fulfill important roles in developmental control and cell-fate decisions. A tremendous amount of progress has accelerated the characterization of histone methylations and the enzymes that regulate them. Deregulation of these histone methyl-modifying enzymes has been increasingly recognized as a hallmark of cancer in the last few years. However, in most cases, we have only limited understanding for the molecular mechanisms by which these enzymes contribute to cancer development and progression. In this review, we summarize the current knowledge regarding some of the best-validated examples of histone lysine methyltransferases and demethylases associated with oncogenesis and discuss their potential mechanisms of action.

Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase.

BACKGROUND: Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. METHODS: A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. RESULTS: Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. CONCLUSIONS: Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.

TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells.

Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation.

Prevalence of Pathogenic Germline BRCA1/2 Variants and Their Association with Clinical Characteristics in Patients with Epithelial Ovarian Cancer in a Rural Area of Japan.

The prevalence of germline BRCA1 or BRCA2 pathogenic variants (gBRCA1/2-PV) in patients with primary epithelial ovarian cancer (OC) in a rural area of Japan and their association with clinical characteristics, including treatment response and survival outcome, were investigated. A total of 123 unbiased patients with OC were tested for gBRCA1 and gBRCA2 using next-generation sequencing-based targeted amplicon sequencing. Clinical characteristics of OC patients with and without gBRCA1/2 status were compared. The overall prevalence of gBRCA1/2-PV was 15.4% (19 cases), with gBRCA2-PV (10.5%, 13 cases) being more common than gBRCA1-PV (4.9%, 6 cases). Among the observed gBRCA1/2-PV, several novel variants were included, suggesting that gBRCA1/2-PV unique to the local area exist. gBRCA1/2-PV was significantly more prevalent in OC patients at an older age, with high-grade serous carcinoma, with advanced-stage tumors, and with a family history of breast cancer or hereditary breast and ovarian cancer syndrome (HBOC)-associated cancers. Patients with advanced-stage OC with gBRCA1/2-PV showed a significantly lower recurrence rate and tended to have better progression-free and overall survival than those with wild-type gBRCA1/2. Genetic testing for gBRCA1/2 status in all OC patients is useful not only for diagnosing HBOC in patients and their relatives to assess the risk of HBOC-associated cancers, but also to estimate therapy response and outcomes in patients.

Dead or alive? Pitfall of survival analysis with TCGA datasets.

We often encounter situations in which data from the TCGA that have been analyzed in papers we read or reviewed cannot be reproduced, even when TCGA datasets are used, especially in survival analyses. Therefore, we attempted to confirm the data source for TCGA survival analysis and found that several websites used to analyze the survival data of TCGA datasets inappropriately handle the survival data, causing differences in statistical analyses. This causes the misinterpretation of results because figures of survival analysis results in several papers are sometimes exactly as generated by these sites, and the results depend on only the tools provided by these sites. We would like to make this situation widely known and raise the problem for scientific soundness.