Long-term culture induces Bax-dependent apoptosis in rat preimplantation embryos.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.

Analysis of Concomitant Medications Prescribed with Antipsychotics to Patients with Dementia.

INTRODUCTION: Antipsychotics are still commonly prescribed to patients with dementia, despite the many issues that have been identified. This study aimed to quantify antipsychotic prescription in patients with dementia and the types of concomitant medications prescribed with antipsychotics. METHODS: A total of 1,512 outpatients with dementia who visited our department between April 1, 2013 and March 31, 2021, were included in this study. Demographic data, dementia subtypes, and regular medication use at the time of the first outpatient visit were investigated. The association between antipsychotic prescriptions and referral sources, dementia subtypes, antidementia drug use, polypharmacy, and prescription of potentially inappropriate medications (PIMs) was evaluated. RESULTS: The antipsychotic prescription rate for patients with dementia was 11.5%. In a comparison of dementia subtypes, the antipsychotic prescription rate was significantly higher for patients with dementia with Lewy bodies (DLB) than for those with all other dementia subtypes. In terms of concomitant medications, patients taking antidementia drugs, polypharmacy, and PIMs were more likely to receive antipsychotic prescriptions than those who were not taking these medications. Multivariate logistic regression analysis showed that referrals from psychiatric institutions, DLB, N-methyl-d-aspartate (NMDA) receptor antagonists, polypharmacy, and benzodiazepine were associated with antipsychotic prescriptions. CONCLUSIONS: Referrals from psychiatric institutions, DLB, NMDA receptor antagonist, polypharmacy, and benzodiazepine were associated with antipsychotic prescriptions for patients with dementia. To optimise prescription of antipsychotics, it is necessary to improve cooperation between local and specialised medical institutions for accurate diagnosis, evaluate the effects of concomitant medication administration, and solve the prescribing cascade.

Asymmetric Strecker reaction at the solid/solid interface.

Related to absolute asymmetric synthesis, a stereospecific reaction at the solid/solid interface arising from crystal chirality of the achiral or racemic substrates has not yet been reported. Here, we demonstrate the asymmetric Strecker-type solid/solid reaction between the chiral crystal of a racemic cyanohydrin (kryptoracemate) and the achiral crystal of an ammonium salt to afford highly enantioenriched α-aminonitrile in combination with amplification of chirality. rac-Cyanohydrin provides its chiral surface as a reactive site and the reaction proceeds with dissociation of cyanohydrin; thus, an asymmetric Strecker-type reaction takes place at the interface of the substrate crystals. Strecker synthesis coupled with cyanohydrin synthesis offers a credible abiotic synthesis mechanism of α-amino acids and α-hydroxy acids. For the first time, stereochemical relationship has been found between the two chiral intermediates, aminonitrile and cyanohydrin, which are in equilibrium in the synthesis mechanism.

Five multicopy gene family genes expressed during the maternal-to-zygotic transition are not essential for mouse development.

Upon fertilization, oocytes transform into totipotent and pluripotent cleavage stage cells through the maternal-to-zygotic transition (MZT), which is regulated by maternal factors and zygotic genome activation (ZGA). Here, we investigated the in vivo function of 16 genes expressed with strong biases in oocytes and cleavage stage embryos by generating knockout (KO) mice. These MZT-associated genes are conserved across many mammalian species and include five multicopy gene family genes: the Nlrp9, Khdc1, Rfpl4, Trim43, and Zscan5 genes. Intercrosses between female KO and male KO mice, including Nlrp9a/b/c triple KO (TKO), Khdc1a/b/c TKO, Rfpl4a/b double KO (DKO), Trim43a/b/c TKO, and Zscan5b KO mice led to the birth to healthy offspring that in turn produced healthy offspring. Our study not only demonstrated that these MZT-associated genes are not essential for mouse development, but also provides valuable resources for analyzing the functions of these genes in other genetic backgrounds, in the presence of stressors, and under pathogenic conditions.

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae.

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.

Relationship between Microsatellite Instability, Immune Cells Infiltration, and Expression of Immune Checkpoint Molecules in Ovarian Carcinoma: Immunotherapeutic Strategies for the Future.

Ovarian cancer has the worst prognosis among gynecological cancers. Thus, new ovarian cancer treatment strategies are needed. Currently, immune checkpoint inhibitors such as anti-PD-1/PD-L1 antibody are attracting attention worldwide. The Food and Drug Administration approved the use of the PD-1 antibody pembrolizumab for solid cancers with microsatellite instability (MSI)-H or mismatch repair (MMR) deficiency in 2017. However, few studies on ovarian carcinoma have evaluated the relationship among MSI status, lymphocyte infiltration into the tumor, and the expression of immune checkpoint molecules by histologic type. We evaluated the expression of MMR proteins, tumor-infiltrating lymphocytes (CD8+), and immune checkpoint molecules (PD-L1/PD-1) by immunohistochemistry in 136 ovarian cancer patients (76, 13, 23, and 24 cases were high-grade serous, mucinous, endometrioid, and clear cell carcinoma, respectively) to investigate the effectiveness of immune checkpoint inhibitors. Only six cases (4.4%) had loss of MMR protein expression. There was no significant relationship between MSI status and age (p = 0.496), FIGO stage (p = 0.357), initial treatment (primary debulking surgery [PDS] or neoadjuvant chemotherapy) (p = 0.419), residual tumor after PDS or interval debulking surgery (p = 0.202), and expression of CD8 (p = 0.126), PD-L1 (p = 0.432), and PD-1 (p = 0.653). These results suggest that only a small number of MSI cases in ovarian cancer can be effectively treated with immune checkpoint inhibitor monotherapy. Therefore, to improve the prognosis of ovarian carcinoma, a combination therapy of immune checkpoint inhibitors and other anticancer drugs is necessary.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems, and their system specificity in mice.

DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.

Exploring new gene integration sites for gene knock-in by gene-trapping strategy.

The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent-positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.

Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.

Prognostic Implication of Three-Dimensional Mitral Valve Tenting Geometry in Dilated Cardiomyopathy.

BACKGROUND AND AIM OF THE STUDY: Functional mitral regurgitation (FMR) is a clinically important complication of left ventricular (LV) dysfunction, occurring as a result of geometric deformity in the mitral valve (MV) complex. The study aim was to determine whether tenting parameters derived from real-time three-dimensional echocardiography (RT3DE) can predict the long-term prognosis for patients with dilated cardiomyopathy (DCM). METHODS: Mitral valve tenting morphology, LV volume and function, and papillary muscle positions were monitored using transthoracic RT3DE in 75 subjects (66 with DCM, nine controls). The maximum tenting sites of the leaflet (maxTS) were also mapped from the reconstructed 3D images, to determine if the 3D tenting parameters correlated to long-term outcome. RESULTS: Follow up information was collected from 62 patients with DCM over a mean period of 42 +/- 31 months. Cardiovascular events occurred in 30 patients (48%), including 13 cardiac deaths (21%). The patients were allocated to an Event group (n = 30) or a Non-event group (n = 32). The LV volumes were significantly larger and LV ejection fraction was lower in the Event group compared to the Non-event group. The 3D tenting volumes were significantly larger in the Event group than the Non- event group (p = 0.05). The maxTS were positioned mostly in the middle portion of the anterior mitral leaflet in the Non-event group (maxTS-mid AML), but in the Event group they were mostly found in the MV coaptation region of the leaflet (maxTS-coapt) (p <0.001). Patients with maxTS-coapt had a worse prognosis compared to those with maxTS-mid AML. On multivariate Cox regression analysis, maxTS was the strongest predictor of event-free survival. CONCLUSION: The 3D tenting pattern, assessed with RT3DE, would be an important clinical parameter in predicting long-term prognosis in patients with DCM.

Cell pairing using microwell array electrodes based on dielectrophoresis.

We report a simple device with an array of 10,000 (100 × 100) microwells for producing vertical pairs of cells in individual microwells with a rapid manipulation based on positive dielectrophoresis (p-DEP). The areas encircled with micropoles which fabricated from an electrical insulating photosensitive polymer were used as microwells. The width (14 µm) and depth (25 µm) of the individual microwells restricted the size to two vertically aligned cells. The DEP device for the manipulation of cells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower microwell array electrode fabricated on an ITO substrate. Mouse myeloma cells stained in green were trapped within 1 s in the microwells by p-DEP by applying an alternating current voltage between the upper ITO and the lower microwell array electrode. The cells were retained inside the wells even after switching off the voltage and washing with a fluidic flow. Other myeloma cells stained in blue were then trapped in the microwells occupied by the cells stained in green to form the vertical cell pairing in the microwells. Cells stained in different colors were paired within only 1 min and a pairing efficiency of over 50% was achieved.

Broadened quantum critical ground state in a disordered superconducting thin film.

A superconductor-insulator transition (SIT) in two dimensions is a prototypical quantum phase transition (QPT) with a clear quantum critical point (QCP) at zero temperature (T = 0). The SIT is induced by a field B and observed in disordered thin films. In some of weakly disordered or crystalline thin films, however, an anomalous metallic (AM) ground state emerges over a wide B range between the superconducting and insulating phases. It remains a fundamental open question how the QPT picture of the SIT is modified when the AM state appears. Here we present measurements of the Nernst effect N, which has great sensitivity to the fluctuations of the superconducting order parameter. From a thorough contour map of N in the B-T plane, we found a thermal-to-quantum crossover line of the superconducting fluctuations, a so-called ghost-temperature line associated with the QPT, as well as a ghost-field line associated with a thermal transition. The QCP is identified as a T = 0 intercept of the ghost-temperature line inside the AM state, which verifies that the AM state is a broadened critical state of the SIT.

Generation of c-Fos knockout rats, and observation of their phenotype.

c-Fos is a useful marker gene of neuron activation for neuroscience and physiology research. The mechanism and function of neural networks have been elucidated using c-Fos reporter knock-in (KI) mice, but the small size of the mice makes it difficult to perform surgical procedures on specific brain regions. On the other hand, there is a large amount of accumulated data on behavioral studies using rats. Thus, the generation of c-Fos reporter rat is expected, but it is difficult to generate gene-modified rats. Furthermore, c-Fos gene abnormality is expected to be severe in rats, as shown in homozygous of c-Fos knockout (KO) mouse, but such analysis has rarely been performed and is not certain. This study generated c-Fos-deficient rats using CRISPR/Cas, with 1067 bp deletion including exon 1 of the c-Fos gene. Homozygous c-Fos KO rats had growth latency and the same tooth and bone abnormality as homozygous c-Fos KO mice but not heterozygous c-Fos KO rats. Therefore, the c-Fos gene in rats is expected to have the same function as that in mice, and the generation of c-Fos reporter KI rats is further anticipated.

Influence of Psychotropic Pro Re Nata Drug Use on Outcomes in Hospitalized Patients with Schizophrenia.

Objective: In the treatment of patients with schizophrenia, pro re nata (PRN) drugs are commonly prescribed for medical indications such as agitation, acute psychiatric symptoms, insomnia, and anxiety. However, high-quality evidence supporting the use of PRN medications is lacking, and these drugs are administered on the basis of clinical experience and habits. Therefore, the actual use of psychotropic PRN drugs and its influence on the patients' outcomes need to be investigated. Methods: This study included 205 patients who underwent inpatient treatment for schizophrenia. We investigated the prescription of psychotropic drugs before admission and at discharge, as well as the dosing frequency of PRN drugs during hospitalization. We also examined the influence of psychotropic PRN drug use on hospitalization days, antipsychotic polypharmacy, and readmission rates. Results: Patients who used psychotropic PRN drugs during hospitalization had significantly longer hospitalization days (p = 7.5 × 10-4) and significantly higher rates of antipsychotic polypharmacy (p = 2.4 × 10-4) at discharge than those who did not use psychotropic PRN drugs. Moreover, a higher number of psychotropic PRN drugs used per day was associated with higher readmission rates within 3 months of discharge (p = 4.4 × 10-3). Conclusion: Psychotropic PRN drug use is associated with prolonged hospitalization, antipsychotic polypharmacy, and increased readmission rates in inpatients with schizophrenia. Therefore, psychiatric symptoms should be stabilized with regularly prescribed medications without the extensive use of psychotropic PRN drugs. Moreover, a system for monitoring and reexamining PRN drug use needs to be established.

Tracing location by applying Emerald luciferase in an early phase of murine endometriotic lesion formation.

The pathogenesis of endometriosis has not been fully elucidated. We focused on the behavior of the ectopic endometrium, that is, the origin of the endometriotic lesion, before adhering to the peritoneal cavity. To observe lesion formation in the very early phase, we developed a novel endometriosis animal model using bioluminescence technology. We established a new transgenic mouse that expressed Emerald luciferase (ELuc) under the control of the CAG promoter. This transgenic mouse, called the CAG-ELuc mouse, showed strong bioluminescence emission; we succeeded in tracing the lesion location by the emission of ELuc. The accuracy of tracing by ELuc was high (57.7-100% of correspondence) and depended on the dosage of E2 administration. In the very early phase after transplantation, the process of lesion formation can be observed non-invasively and chronologically. We have verified that the preferred location of the uterus (transplanted grafts) was fixed immediately after the transplantation of the grafts.

Clinical Outcomes of Genotype-Matched Therapy for Recurrent Gynecological Cancers: A Single Institutional Experience.

Recent advances in next-generation sequencing and genome medicine have contributed to treatment decisions in patients with cancer. Most advanced gynecological cancers develop resistance to chemotherapy and have a poor prognosis. Therefore, we conducted genomic tests in gynecological tumors to examine the efficacy and clinical feasibility of genotype-matched therapy. Target sequencing was performed in 20 cases of gynecological cancers (cervical cancer, 6; endometrial cancer, 6; and ovarian cancer, 6). Both actionable and druggable genes were identified in 95% (19/20) of the cases. Among them, seven patients (35%) received genotype-matched therapy, which was effective in three patients. Of the three patients, one patient with a PTEN mutation received everolimus, another patient with a TSC2 mutation received everolimus and letrozole, and the patient with a BRIP1 mutation received olaparib. Subsequently, disease control in these three patients lasted for more than half a year. However, all patients relapsed between 9 and 13 months after the initiation of genotype-matched therapy. In this study, the response rate of genotype-matched therapy was 43% (3/7), which may have contributed to improved prognoses. Therefore, genotype-matched therapies may help patients with refractory gynecological cancers achieve better outcomes.

Single-port laparoscopic surgery for ovarian cystectomy: A single-center analysis of 25 cases.

Innovation in gynecological surgery is constantly evolving to make procedures less invasive. Minimally invasive single-port laparoscopic surgery (SPLS) is another innovation that may further improve gynecological surgery outcomes. However, SPLS is not widely used due to the technical difficulties of the procedure. Inserting several instruments through the same incision impedes proper use of the devices. Therefore, the present study aimed to find a technique to overcome this problem and make this approach more convenient. Between March 2015 and February 2020, 25 patients were treated with SPLS by a single gynecological surgeon. The range of surgery time was 50-103 min and the mean surgery time was 67.2 min. The mean bleeding volume was small (mean, 10.1 ml). No intraoperative or postoperative complications occurred in the patients. A gel port (GelPOINT® Mini Medical Leaders) was inserted into the peritoneal cavity through a 3-cm Z-shaped intra-umbilical skin incision. Additionally, a small incision (3 mm) was made in the left medial portion of the iliac crest and a bladeless trocar (Versa One®) was inserted. Thus, crowding of the working instruments within one incision was prevented. The addition of a small diameter port (3-mm) at the wound site left practically no scar, thus making SPLS a cosmetically superior option compared with a bigger diameter port (5-mm).

P16INK4A expression might be associated with a favorable prognosis for cervical adenocarcinoma via dysregulation of the RB pathway.

Previous studies have largely failed to clarify the relationship between p16INK4A status and cervical adenocarcinoma prognosis. The current study aimed to examine the clinical and pathological significance of p16INK4A expression in several cervical adenocarcinoma subtypes. Eighty-two samples collected from patients with cervical adenocarcinoma were formalin fixed and paraffin embedded. Next, p16INK4A levels were analyzed with immunohistochemistry. Additionally, the relationship between p16INK4A expression and clinicopathological factors as well as prognosis was evaluated. The expression of p16INK4A was mostly detected in all usual cervical adenocarcinoma subtypes. In the gastric type, only a few cases were positive for p16INK4A expression. Results of the Kaplan-Meier analysis indicated that the positive p16INK4A expression in tumor cells was significantly associated with favorable progression-free survival and overall survival in patients with cervical adenocarcinoma (p = 0.018 and p = 0.047, respectively, log-rank test). Our findings suggest that the status of p16INK4A expression may influence prognosis. Thus, p16INK4A expression could be used as a biomarker for improving the prognosis of patients with cervical adenocarcinoma.

Intratumoral Genomic Heterogeneity May Hinder Precision Medicine Strategies in Patients with Serous Ovarian Carcinoma.

Precision medicine, which includes comprehensive genome sequencing, is a potential therapeutic option for treating high-grade serous carcinoma (HGSC). However, HGSC is a heterogeneous tumor at the architectural, cellular, and molecular levels. Intratumoral molecular heterogeneity currently limits the precision of medical strategies based on the gene mutation status. This study was carried out to analyze the presence of 160 cancer-related genetic alterations in three tissue regions with different pathological features in a patient with HGSC. The patient exhibited histological heterogeneous features with different degrees of large atypical cells and desmoplastic reactions. TP53 mutation, ERBB2 and KRAS amplification, and WT1, CDH1, and KDM6A loss were detected as actionable gene alterations. Interestingly, the ERBB2 and KRAS amplification status gradually changed according to the region examined. The difference was consistent with the differences in pathological features. Our results demonstrate the need for sampling of the appropriate tissue region showing progression of pathological features for molecular analysis to solve issues related to tumor heterogeneity prior to developing precision oncology strategies.