Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genome-wide analysis of histone modifications can contribute to the identification of candidate cis-regulatory regions in the threespine stickleback fish.

BACKGROUND: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq). RESULTS: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions. CONCLUSIONS: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

Availability of genome-matched therapy based on clinical practice.

BACKGROUND: Comprehensive genomic profiling (CGP) provides new opportunities for patients with advanced cancer to receive genome-matched therapies, but the availability rate of these remains low. We reviewed our CGP cases and suggested possible strategies to improve the current status from a clinical perspective. METHODS: Druggable genomic alterations and barriers to accessing genome-matched therapies were investigated in 653 patients with 30 various types of cancers who underwent CGP. RESULTS: While the availability rate of genome-matched therapies as a whole was 9.5%, CGP was useful in some cancer types. Patients with thyroid cancer and lung cancer harbored druggable genomic alterations at high rates, while sarcoma rarely harbored these alterations (100%, 76%, and 15.2%, respectively). In contrast, the availability rate of genome-matched therapies was highest in patients with sarcoma and head and neck cancer (HNC) (60% and 40%, respectively). One hundred thirteen patients (63.5%) had multiple barriers to accessing genome-matched therapy. Of 178 patients, 21 patients (11.8%) could not be considered for genome-matched therapies solely because of the deterioration of their performance status. CONCLUSION: This study demonstrated the usefulness of CGP for patients with sarcoma and HNC in addition to lung cancer in clinical practice. Performing CGP at the front line has the potential to improve the availability of genome-matched therapy.

Prognostic impact of cancer genomic profile testing for advanced or metastatic solid tumors in clinical practice.

Cancer genomic profile (CGP) testing, which is covered by the national health insurance system in Japan, has been introduced as a routine clinical practice. However, the effects of CGP testing on prognoses remain unclear. Drug accessibility rates and prognoses after CGP testing were retrospectively investigated in 713 patients who underwent CGP testing examined by our molecular tumor board between November 2019 and October 2022,. Overall survival (OS) was examined using the log-rank test and the Kaplan-Meier method. The median age of patients (326 males and 387 females) was 58 years (12-85 years). CGP testing revealed one or more gene mutations in 681 cases (95.5%), among which actionable gene mutations were detected in 439 (61.6%). Although treatment options were recommended for 285 cases (40.0%) by the molecular tumor board, only 45 received treatment based on their gene mutations. During the median observation period of 8.6 months, 351 (49.2%) patients died of the exacerbation of existing diseases. No significant differences were observed in OS between patients treated with and without genomically matched therapy (p = 0.285). According to clinical responses to treatment based on gene mutations, median OS was significantly longer in patients who achieved partial response and stable disease (26.5 months; 95% CI 14.4-38.6) than in those with progressive disease and not evaluated (9.8 months; 95% CI 5.8-13.8, p = 0.013). Responses to treatment based on gene mutations may improve prognoses, and it is important to increase the drug accessibility rate after CGP testing.

Chromatin profile-based identification of a novel ER-positive breast cancer subgroup with reduced ER-responsive element accessibility.

BACKGROUND: Oestrogen receptor (ER) signalling-dependent cancer cell growth is one of the major features of ER-positive breast cancer (BC). Inhibition of ER function is a standard and effective treatment for ER-positive tumours; however, ~20% of patients with ER-positive BC experience early or late recurrence. In this study, we examined intertumour heterogeneity from an epigenetic perspective based on the hypothesis that the intrinsic difference in epigenetic states around ER signalling pathway underlies endocrine therapy resistance. METHODS: We performed transposase-accessible chromatin sequencing (ATAC-seq) analysis of 42 BC samples, including 35 ER-positive(+) human epidermal growth factor receptor 2 (HER2)-negative(-) and 7 triple-negative tumours. We also reanalysed ATAC-seq data of 45 ER + /HER2 - tumours in the Cancer Genome Atlas (TCGA) BC cohort to validate our observations. RESULTS: We conducted a comprehensive analysis of cis-regulatory elements (CREs) using ATAC-seq, identifying three subgroups based on chromatin accessibility profiles. We identified a subgroup of ER-positive BCs with a distinctive chromatin accessibility pattern including reduced accessibility to ER-responsive elements (EREs). The same subgroup was also observed in TCGA BC cohort. Despite the reduced accessibility to EREs, the expression of ER and potential ER target genes were not decreased in these tumours. CONCLUSION: Our findings highlight the existence of a subset of ER-positive BCs with unchanged ER expression but reduced EREs accessibility that cannot be distinguished by conventional immunostaining for ER. Future studies should determine whether these tumours are associated with resistance to endocrine therapy.

Genomic features of Helicobacter pylori-naïve diffuse-type gastric cancer.

Helicobacter pylori (HP) is a major etiologic driver of diffuse-type gastric cancer (DGC). However, improvements in hygiene have led to an increase in the prevalence of HP-naïve DGC; that is, DGC that occurs independent of HP. Although multiple genomic cohort studies for gastric cancer have been conducted, including studies for DGC, distinctive genomic differences between HP-exposed and HP-naïve DGC remain largely unknown. Here, we employed exome and RNA sequencing with immunohistochemical analyses to perform binary comparisons between 36 HP-exposed and 27 HP-naïve DGCs from sporadic, early-stage, and intramucosal or submucosal tumor samples. Among the samples, 33 HP-exposed and 17 HP-naïve samples had been preserved as fresh-frozen samples. HP infection status was determined using stringent criteria. HP-exposed DGCs exhibited an increased single nucleotide variant burden (HP-exposed DGCs; 1.97 [0.48-7.19] and HP-naïve DGCs; 1.09 [0.38-3.68] per megabase; p = 0.0003) and a higher prevalence of chromosome arm-level aneuploidies (p < 0.0001). CDH1 was mutated at similar frequencies in both groups, whereas the RHOA-ARHGAP pathway misregulation was exclusive to HP-exposed DGCs (p = 0.0167). HP-exposed DGCs showed gains in chromosome arms 8p/8q (p < 0.0001), 7p (p = 0.0035), and 7q (p = 0.0354), and losses in 16q (p = 0.0167). Immunohistochemical analyses revealed a higher expression of intestinal markers such as CD10 (p < 0.0001) and CDX2 (p = 0.0002) and a lower expression of the gastric marker, MUC5AC (p = 0.0305) among HP-exposed DGCs. HP-naïve DGCs, on the other hand, had a purely gastric marker phenotype. This work reveals that HP-naïve and HP-exposed DGCs develop along different molecular pathways, which provide a basis for early detection strategies in high incidence settings. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genomic determinants impacting the clinical outcome of mogamulizumab treatment for adult T-cell leukemia/lymphoma.

In order to identify genomic biomarkers for the outcome of mogamulizumab-containing treatment, an integrated molecular analysis of adult T-cell leukemia/lymphoma (ATL) was conducted on 64 mogamulizumab-naïve patients. Among driver genes, CCR4 and CCR7 alterations were observed in 22% and 11% of the patients, respectively, both consisting of single nucleotide variants (SNV)/insertion-deletions (indels) in the C-terminus. Patients with CCR4 alterations or without CCR7 alterations exhibited a more favorable clinical response (complete response [CR] rate 93%, 13/14; P=0.024, and CR rate 71%, 40/56; P=0.036, respectively). Additionally, TP53, CD28, and CD274 alterations were identified in 35%, 16%, and 10% of the patients, respectively. TP53 alterations included SNV/indels or copy number variations (CNV) such as homozygous deletion; CD28 alterations included SNV, CNV such as amplification, or fusion; CD274 alterations included CNV such as amplification, or structural variants. Univariate analysis revealed that TP53, CD28 or CD274 alterations were associated with worse overall survival (OS) (hazard ratio [HR]: 2.330, 95% confidence interval [CI]: 1.183-4.589; HR: 3.191, 95% CI: 1.287- 7.911; HR: 3.301, 95% CI: 1.130-9.641, respectively) but that CCR4 alterations were associated with better OS (HR: 0.286, 95% CI: 0.087-0.933). Multivariate analysis indicated that in addition to performance status, TP53, CCR4 or CD274 alterations (HR: 2.467, 95% CI: 1.197-5.085; HR: 0.155, 95% CI: 0.031-0.778; HR: 14.393, 95% CI: 2.437-85.005, respectively) were independently and significantly associated with OS. The present study contributes to the establishment of precision medicine using mogamulizumab in ATL patients.

Immunogenic characteristics of microsatellite instability-low esophagogastric junction adenocarcinoma based on clinicopathological, molecular, immunological and survival analyses.

Microsatellite instability (MSI) is categorized by mutation frequency: high MSI (MSI-H), low MSI (MSI-L) and microsatellite stable (MSS). MSI-H tumors have a distinct immunogenic phenotype, with immunotherapies using checkpoint inhibitors already approved for the treatment of MSI-H gastroesophageal adenocarcinoma (GEA); this is not observed for MSI-L or MSS. Here, we tested the hypothesis that MSI-L tumors are also a distinct phenotype and potentially immunogenic. MSI-PCR assays (BAT25, BAT26, BAT40, D2S123, D5S346 and D17S250) were performed on 363 Epstein-Barr virus-negative, surgically resected esophagogastric junction (EGJ) adenocarcinoma samples. Tumors were characterized as MSI-H (≥2 markers), MSI-L (1 marker) or MSS (0 markers). CD8+ cell counts, PD-L1 and HER2 expression levels, TP53 mutations, epigenetic alterations and prognostic significance were also examined. All pathological and molecular experiments were conducted using serial, whole-tumor sections of chemo-naïve surgical specimens. MSI-H and MSI-L were assigned to 28 (7.7%) and 24 (6.6%) cases, respectively. Compared to MSS cases, MSI-L cases had significantly higher intratumoral CD8+ cell infiltration (P = .048) and favorable EGJ cancer-specific survival (multivariate hazard ratio = 0.35, 95% CI, 0.12-0.82; P = .012). MSI-L tumors were also significantly associated with TP53-truncating mutations as compared to MSI-H (P = .009) and MSS (P = .012) cases, and this trend was also observed in GEA data from The Cancer Genome Atlas (TCGA). Indel mutational burden among TCGA MSI-L tumors was significantly higher than that of MSS tumors (P = .016). These results suggest that MSI-L tumors may have a distinct tumor phenotype and be potentially immunogenic in EGJ adenocarcinoma.

Integrative genome-wide analyses reveal the transcriptional aberrations in Japanese esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease. At present, the genomic profiles of ESCC are known to a considerable extent, and DNA methylation and gene expression profiles have been mainly used for the classification of ESCC subtypes, but integrative genomic, transcriptomic, and epigenomic analyses remain insufficient. Therefore, we performed integrative analyses using whole-exome sequencing, DNA methylation, and RNA sequencing (RNA-seq) analyses of Japanese patients with ESCC. In cancer-related genes, such as NOTCH family genes, RTK/PI3K pathway genes, and NFE2L2 pathway genes, variants and copy number amplification were detected frequently. Japanese ESCC cases were clustered into two mutational signatures: an APOBEC-associated signature and an age-related signature. In imprinted genes, DNA methylation was aberrant in gene promoter regions and correlated well with gene expression profiles. Nonsynonymous single-nucleotide variants and allelic expression imbalance were detected frequently in FAT family genes. Our integrative genome-wide analyses, including DNA methylation and allele-specific gene expression profiles, revealed altered gene regulation of imprinted genes and FAT family genes in ESCC.

Pathogenicity assessment of variants for breast cancer susceptibility genes based on BRCAness of tumor sample.

Genes involved in the homologous recombination repair pathway-as exemplified by BRCA1, BRCA2, PALB2, ATM, and CHEK2-are frequently associated with hereditary breast and ovarian cancer syndrome. Germline mutations in the loci of these genes with loss of heterozygosity or additional somatic truncation at the WT allele lead to the development of breast cancers with characteristic clinicopathological features and prominent genomic features of homologous recombination deficiency, otherwise referred to as "BRCAness." Although clinical genetic testing for these and other genes has increased the chances of identifying pathogenic variants, there has also been an increase in the prevalence of variants of uncertain significance, which poses a challenge to patient care because of the difficulties associated with making further clinical decisions. To overcome this challenge, we sought to develop a methodology to reclassify the pathogenicity of these unknown variants using statistical modeling of BRCAness. The model was developed with Lasso logistic regression by comparing 116 genomic attributes derived from 37 BRCA1/2 biallelic mutant and 32 homologous recombination-quiescent breast cancer exomes. The model showed 95.8% and 86.7% accuracies in the training cohort and The Cancer Genome Atlas validation cohort, respectively. Through application of the model for variant reclassification of homologous recombination-associated hereditary breast and ovarian cancer causal genes and further assessment with clinicopathological features, we finally identified one likely pathogenic and five likely benign variants. As such, the BRCAness model developed from the tumor exome was robust and provided a reasonable basis for variant reclassification.

T-cell complexity and density are associated with sensitivity to neoadjuvant chemoradiotherapy in patients with rectal cancer.

Emerging evidence suggests that an increased density of pre-treatment CD8+ tumor-infiltrating lymphocytes (TILs) is associated with good response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. However, the significance of T-cell complexity in the clinical setting remains unknown. High-throughput T-cell receptor (TCR) ß sequencing was applied to quantify the TCR repertoire of pre-treatment biopsies from 67 patients with advanced rectal cancer receiving preoperative CRT. Diversity index was used to represent the complexity of the TCR repertoire in a tumor. Pre-treatment CD8+ TIL densities were assessed by immunohistochemistry. Changes in TCR repertoire before and after CRT were also analysed in 23 patients. Diversity indices were significantly higher for good responders than for non-responders (P = 0.031). The multivariate analysis revealed that both CD8+ TIL density and TCR diversity index were independently associated with good response to CRT (P < 0.001 and P = 0.049, respectively). Patients who were high for both CD8+ TIL density and TCR diversity (double-high) had markedly better responses to CRT than double-low patients (84.2% vs 16.7%, P < 0.0001). Larger changes in TCR repertoires before and after CRT were correlated with better recurrence-free survival (P = 0.027). The complexity and dynamic change in the TCR repertoire might serve as a useful indicator of response to CRT in combination with CD8+ TIL density in patients with rectal cancer.

Clinicopathological and genomic features in patients with head and neck neuroendocrine carcinoma.

Neuroendocrine carcinoma (NEC) of the head and neck is a rare type of malignancy, accounting for only 0.3% of all head and neck cancers, and its clinicopathological and genomic features have not been fully characterized. We conducted a retrospective analysis of 27 patients with poorly differentiated NEC of the head and neck seen at our institution over a period of 15 years. Patient characteristics, adopted therapies, and clinical outcomes were reviewed based on the medical records. Pathological analysis and targeted sequencing of 523 cancer-related genes were performed using evaluable biopsied/resected specimens based on the clinical data. The most common tumor locations were the paranasal sinus (33%) and the oropharynx (19%). Eighty-one percent of the patients had locally advanced disease. The 3-year overall survival rates in all patients and in the 17 patients with locally advanced disease who received multimodal curative treatments were 39% and 53%, respectively. Histologically, large cell neuroendocrine carcinoma was the predominant subtype (58% of evaluable cases), and the Ki-67 labeling index ranged from 59 to 99% (median: 85%). Next-generation sequencing in 14 patients identified pathogenic/likely pathogenic variants in TP53, RB1, PIK3CA-related genes (PREX2, PIK3CA, and PTEN), NOTCH1, and SMARCA4 in six (43%), three (21%), two (14%), two (14%), and one (7%) patients, respectively. Sequencing also detected the FGFR3-TACC3 fusion gene in one patient. The median value of the total mutational burden (TMB) was 7.1/Mb, and three patients had TMB ≥ 10. Regardless of the aggressive pathological features, our data revealed favorable clinical characteristics in the patients with locally advanced disease who received curative treatment. The lower TP53 and RB1 mutation prevalence rates compared to those described for small cell lung cancer suggests the biological heterogeneity of NEC in different parts of the body. Furthermore, the FGFR3-TACC3 fusion gene and mutations in genes encoding the components of the NOTCH and PI3K/AKT/mTOR pathways found in our study may be promising targets for NEC of the head and neck.

Two Distinct Tumorigenic Processes in Endometrial Endometrioid Adenocarcinoma.

Endometrial endometrioid adenocarcinoma (EEA) is conventionally considered to be a single pathologic entity that develops through a hyperplasia-carcinoma sequence under the influence of estrogen. Previously, another EEA subtype was described and proposed to arise directly from normal endometrium. These conventional and de novo subtypes are designated groups 1 and 2, respectively. To identify the molecular mechanisms of these distinct tumorigenic processes, we conducted comprehensive integrated analyses of genomic data with hormonal status for group 1 paired carcinoma and hyperplasia and group 2 carcinoma samples. Although group 1 carcinomas mostly exhibited genomically stable characteristics and the activation of estrogen signaling, group 2 EEAs showed enriched hypermutator and CpG island methylator phenotypes. Pairwise comparisons of hyperplasia and carcinoma, along with time-course analyses of the hyperplasia-carcinoma sequence, revealed the acquisition of driver mutations in the evolutionary process of hyperplasia but not in the transition from hyperplasia to carcinoma. The current study confirms the existence of two different histopathologic programs during EEA development that harbor distinct molecular bases and demonstrates the biological relevance of these differential tumorigenic processes.

Genomic alterations in gynecological malignancies: histotype-associated driver mutations, molecular subtyping schemes, and tumorigenic mechanisms.

There are numerous histological subtypes (histotypes) of gynecological malignancies, with each histotype considered to largely reflect a feature of the "cell of origin," and to be tightly linked with the clinical behavior and biological phenotype of the tumor. The recent advances in massive parallel sequencing technologies have provided a more complete picture of the range of the genomic alterations that can persist within individual tumors, and have highlighted the types and frequencies of driver-gene mutations and molecular subtypes often associated with these histotypes. Several large-scale genomic cohorts, including the Cancer Genome Atlas (TCGA), have been used to characterize the genomic features of a range of gynecological malignancies, including high-grade serous ovarian carcinoma, uterine corpus endometrial carcinoma, uterine cervical carcinoma, and uterine carcinosarcoma. These datasets have also been pivotal in identifying clinically relevant molecular targets and biomarkers, and in the construction of molecular subtyping schemes. In addition, the recent widespread use of clinical sequencing for the more ubiquitous types of gynecological cancer has manifested in a series of large genomic datasets that have allowed the characterization of the genomes, driver mutations, and histotypes of even rare cancer types, with sufficient statistical power. Here, we review the field of gynecological cancer, and seek to describe the genomic features by histotype. We also will demonstrate how these are linked with clinicopathological attributes and highlight the potential tumorigenic mechanisms.

Fine-scale genetic structure of the endangered bitterling in the middle river basin of the Kiso River, Japan.

Recently, anthropogenic alterations have had severe and negative impacts on the terrestrial and aquatic species and environments. To conserve species that have a small and limited habitat, it is necessary to focus on fine-scale population structure and its effects on persistence. The deepbodied bitterling Acheilognathus longipinnis is an endangered freshwater fish that occupies ponds scattered in lateral bars in the Kiso River. In this study, we conducted multi-locus microsatellite DNA analysis to evaluate both fine-scale population structure and genetic diversity, in order to conserve A. longipinnis. The smaller number of loci deviating from the Hardy-Weinberg equilibrium in ponds scattered in individual lateral bars compared to the whole river system suggests that A. longipinnis forms a local breeding population in units of ponds. The population was roughly split between the river banks and the local population located in ponds in the mid-channel bar showed intermediate relationships with the river bank populations. Gene flow between local populations was not always homogeneous and was not influenced by geographical distances between local populations or the direction of river flow. The dispersal of A. longipinnis across both river bank sides may be constrained and is probably affected by the ecological characteristics of A. longipinnis and the hydrological regimes. Consequently, A. longipinnis in the Kiso River is maintained as a complex of multiple local populations with appropriate gene flow among them. To conserve A. longipinnis, both the persistence of the unstable ponds and moderate genetic exchanges by individual migration are required.

Immunogenomic landscape of gynecologic carcinosarcoma.

OBJECTIVE: Carcinosarcoma (CS) of the uterus or ovary is a rare, biphasic tumor comprising epithelial and mesenchymal elements, and exhibits more aggressive clinical features than its carcinoma counterpart. Four molecular subtypes of CS were recently established based on genomic aberration profiles (POLE, MSI, CNH, and CNL) and shown to be associated with multiple clinicopathological parameters, including patient outcomes. However, the role of the immune microenvironment in CS remains unclear. Here, we investigated the influence of the immune cells that infiltrate CS to better understand the immunological status of gynecological CS. METHODS: Tumor immune microenvironmental analyses on CS samples were performed using immune cell profiling with RNA-seq, transcriptomic subtyping with microenvironmental genes, and T-cell receptor repertoire assay. Carcinoma and sarcoma elements from CS samples were also assessed separately. RESULTS: Relying on estimations of tumor-infiltrating cell types from RNA-seq data, POLE and MSI (hypermutator) tumors showed an enrichment of M1 macrophages, plasma cells and CD8+ T cells, whereas CNH and CNL (non-hypermutator) tumors had high levels of M2 macrophages. Further subclassification by immune-related, non-cancer genes identified a fraction of tumors with distinct patient outcomes, particularly those with the CNH genomic aberration subtype. T-cell heterogeneity was independently correlated with prolonged progression-free survival. Differential analysis of carcinoma and sarcoma elements identified many shared mutations but there was little overlap in the T-cell receptor repertoire between the two elements. CONCLUSIONS: Tumor immune microenvironmental analyses could offer potential clinical utility in the stratification of gynecological CS above classification by genomic aberration subtype alone.

Multiple waves of freshwater colonization of the three-spined stickleback in the Japanese Archipelago.

BACKGROUND: The three-spined stickleback (Gasterosteus aculeatus) is a remarkable system to study the genetic mechanisms underlying parallel evolution during the transition from marine to freshwater habitats. Although the majority of previous studies on the parallel evolution of sticklebacks have mainly focused on postglacial freshwater populations in the Pacific Northwest of North America and northern Europe, we recently use Japanese stickleback populations for investigating shared and unique features of adaptation and speciation between geographically distant populations. However, we currently lack a comprehensive phylogeny of the Japanese three-spined sticklebacks, despite the fact that a good phylogeny is essential for any evolutionary and ecological studies. Here, we conducted a phylogenomic analysis of the three-spined stickleback in the Japanese Archipelago. RESULTS: We found that freshwater colonization occurred in multiple waves, each of which may reflect different interglacial isolations. Some of the oldest freshwater populations from the central regions of the mainland of Japan (hariyo populations) were estimated to colonize freshwater approximately 170,000 years ago. The next wave of colonization likely occurred approximately 100,000 years ago. The inferred origins of several human-introduced populations showed that introduction occurred mainly from nearby habitats. We also found a new habitat of the three-spined stickleback sympatric with the Japan Sea stickleback (Gasterosteus nipponicus). CONCLUSIONS: These Japanese stickleback systems differ from those in the Pacific Northwest of North America and northern Europe in terms of divergence time and history. Stickleback populations in the Japanese Archipelago offer valuable opportunities to study diverse evolutionary processes in historical and contemporary timescales.

Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification.

Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated ß-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.