Cancer Science

The development of mesothelin (MSLN) epitope reactive T cells is observed in mice that are immunized with the MSLN vaccine. Engineered T cells expressing MSLN‐reactive high‐affinity TCR exhibit extraordinary therapeutic effects for invasive pancreatic ductal adenocarcinoma in a mouse model. However, the generation of MSLN‐reactive T cells through the introduction of MSLN‐deficient thymus and the transplantation of the latter as a cure for cancer treatment have not been tested to date. In the present study, the expression of MSLN was mainly identified in medullary thymic epithelial cells (mTECs) but not in hematopoietic cells, cortical thymic epithelial cells (cTECs), endothelial cells, or fibroblast cells in the thymus. The increasement of activated T cells was observed in MSLN‐expressing tumors from MSLN‐deficient mice, indicating that MSLN‐reactive T cells had developed. Finally, in an AOM‐DSS‐induced mouse model of colorectal cancer (CRC), transplantation of MSLN‐deficient thymus repressed the progression of CRC, accompanied by an increased number of IFNγ‐expressing T lymphocytes in the tumors. The data from this study demonstrated that ectopic transplantation of MSLN‐deficient thymus induced MSLN‐specific antitumor responses to MSLN‐expressing tumors, and thus attenuated tumor progression.

DNA methylation is an enzyme‐driven epigenetic modification that must be precisely regulated to maintain cellular homeostasis. Aberrant methylation status, especially hypermethylation of the promoter sites of tumor‐suppressor genes, is observed in human malignancies and is a proven target for cancer therapy. The first‐generation DNA demethylating agents, azacitidine and decitabine, are widely used for treating several hematological malignancies. In addition, orally bioavailable prodrugs of azacitidine and decitabine have recently been approved by the FDA. We have developed a silylated derivative of decitabine, OR‐2100, which is resistant to degradation by cytidine deaminase and orally bioavailable. It has efficacy against several human hematological malignancies in xenograft mouse models with less hematotoxicity than decitabine. Since DNA demethylating agents are combined with molecularly targeted drugs in clinical use and trials, we think that the less hematotoxic profile of OR‐2100 makes it suitable for use as a combination therapy. In this article, we review the therapeutic approach in hematological malignancies with the DNA demethylating agent OR‐2100.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis and limited treatment options. While the majority of PDAC cases harbor KRAS mutations, approximately 8%–10% are KRAS wild‐type (KRAS‐WT). These KRAS‐WT tumors often contain actionable mutations and gene fusions, making them more suitable for precision therapies. Identifying these molecular alterations is crucial for improving outcomes in this subset of patients. This retrospective study involved 34 patients with KRAS‐WT PDAC. Genomic profiling was performed using next‐generation sequencing (NGS) and RNA sequencing to detect mutations and fusions. Comparative analysis was conducted with TCGA‐PAAD data, and immune infiltration was assessed using bioinformatic deconvolution methods. Targetable alterations were identified in multiple pathways. Key mutations included ATM (18%), PIK3CA (15%), and ROS1 (15%), while actionable gene fusions such as CCDC6‐RET and ETV6‐NTRK3 were present in 10.3% of patients. The gene mutations associated with homologous recombination deficiency (HRD) are predicted to increase sensitivity to platinum‐based chemotherapy (p = 0.047). Tumors with epigenetic regulatory genes mutations (e.g., ARID1A, KMT2C/D) exhibited enhanced immune cell infiltration, highlighting potential responsiveness to immune checkpoint inhibitors (ICIs). Kinase fusions (NTRK and RET) were linked to response to larotinib and RET‐specific inhibitors, respectively. KRAS‐WT PDAC contains actionable mutations and fusions, offering significant potential for targeted and immune‐based therapies. Further clinical studies are needed to validate these therapeutic approaches.

To report clinical outcomes following highly hypofractionated biaxially rotational dynamic radiation therapy (BROAD‐RT), a unique radiation therapy method that facilitates non‐coplanar volumetric‐modulated arc therapy (VMAT) without the need to rotate the couch or reposition the patient, for high‐risk prostate cancer (PCa) with simultaneous integrated boost (SIB) for intra‐prostatic dominant lesions (IPDLs), we performed a single‐center prospective pilot study. In this study, patients with high‐risk PCa according to the D'Amico classification or those with cT3aN0M0 PCa were eligible. VMAT was performed using BROAD‐RT, and a dose of 54 Gy in 15 fractions was prescribed for the prostate in combination with SIB for IPDLs at a dose of 57 Gy in 15 fractions. Short‐term neoadjuvant androgen‐deprivation therapy (median: 6.9 months) was conducted. Neither adjuvant androgen‐deprivation therapy nor fiducial marker implantation to the prostate was applied for any patient. In total, 26 patients were registered in this study between August 2018 and November 2020. Their median age was 73 years at the initiation of RT. The median follow‐up period was 49.7 months. The 4‐year cumulative incidence rates of grade 2 late GU and GI toxicities were 15.4 and 3.8%, respectively. No grade 3 or higher acute or late toxicities were observed. The 4‐year biochemical failure‐free survival rates were 87.7%. In conclusion, highly hypofractionated RT using BROAD‐RT for high‐risk PCa with SIB for IPDLs was feasible and facilitated favorable oncological outcomes. Therefore, this approach is considered a promising method to achieve safe dose escalation and shorten the treatment duration.

CBA‐1205 is a novel humanized antibody targeting delta‐like 1 homolog (DLK1) that enhances antibody‐dependent cellular cytotoxicity activity. DLK1 overexpression has been reported in various cancer types, such as hepatocellular carcinoma and neuroblastoma. CBA‐1205 demonstrates potent antitumor activity in multiple tumor models, making it a potential treatment option for DLK1‐expressing cancers. This first‐in‐human, open‐label Phase I study includes three parts. Part 1, the dose‐escalation phase, primarily evaluates the safety profile, tolerability, and maximum tolerated dose of CBA‐1205. The drug is administered intravenously every 2 weeks in a 28‐day cycle. A standard 3 + 3 dose‐escalation design was used across seven cohorts. In a cohort of 22 Japanese patients, over 80% had undergone three or more prior treatments. CBA‐1205 was well tolerated, with no dose‐limiting toxicity observed at doses ranging from 0.1 to 30 mg/kg, the planned highest dose. There were no treatment‐related serious adverse events or trial‐related deaths. CBA‐1205 exposure, as measured by Cmax, AUC0–14, and AUC0–∞, increased in a dose‐dependent manner. No serum anti‐CBA‐1205 antibodies were detected. Serum DLK1 concentrations were found in 6 out of 22 patients. Stable disease for over 6 months was observed in six patients, with progression‐free survival ranging from 29 to 144 weeks. CBA‐1205 was well tolerated, showing no severe toxicity in patients with advanced or recurrent solid tumors. The favorable safety profile and indications of potential activity support further investigation in Parts 2 and 3 of this Phase I study to evaluate the safety, tolerability, and preliminary efficacy of CBA‐1205.

Colorectal cancer (CRC) is well characterized in terms of genetic mutations and the mechanisms by which they contribute to carcinogenesis. Mutations in APC, TP53, and KRAS are common in CRC, indicating key roles for these genes in tumor development and progression. However, for certain tumors with low frequencies of these mutations that are defined by tumor location and molecular phenotypes, a carcinogenic mechanism dependent on BRAF mutations has been proposed. We here analyzed targeted sequence data linked to clinical information for CRC, focusing on tumors with a high tumor mutation burden (TMB) in order to identify the characteristics of associated mutations, their relations to clinical features, and the mechanisms of carcinogenesis in tumors lacking the major driver oncogenes. Analysis of overall mutation frequencies confirmed that APC, TP53, and KRAS mutations were the most prevalent in our cohort. Compared with other tumors, TMB‐high tumors were more frequent on the right side of the colon, had lower KRAS and higher BRAF mutation frequencies as well as a higher microsatellite instability (MSI) score, and showed a greater contribution of a mutational signature associated with MSI. Ranking of variant allele frequencies to identify genes that play a role early in carcinogenesis suggested that mutations in genes related to the DNA damage response (such as ATM and POLE) and to MSI (such as MSH2 and MSH6) may precede BRAF mutations associated with activation of the serrated pathway in TMB‐high tumors. Our results thus indicate that TMB‐high tumors suggest that mutations of genes related to mismatch repair and the DNA damage response may contribute to activation of the serrated pathway in CRC.

Angiosarcoma (AS) is a rare, aggressive malignancy originating from vascular or lymphatic endothelial cells. Despite its severity, little is known about its epidemiology, and no geographical regions have previously been identified as having an exceptionally high incidence. We retrospectively analyzed medical records spanning 37 years (1987–2023) in Okinawa, Japan, identifying 135 cases of AS that were used to calculate its incidence. This incidence was compared to global data to highlight significant regional differences. Factors related to patients' survival were also assessed. The age‐adjusted incidence of AS of the scalp in Okinawa was 4.1 per million per year (mpy; 2015 Japanese model population) or 2.0 per mpy (2000 US standard population), significantly higher than the global data, including in the United States (about eightfold higher) and mainland Japan (about fourfold higher). The estimated five‐year survival for patients with AS of the scalp in Okinawa was 9.2%. Multivariate analysis identified surgery, chemotherapy, and radiotherapy as significant factors associated with patient survival. This study provides the first evidence of a significantly higher incidence rate of AS of the scalp in Okinawa. Given its rarity, further research is crucial to uncover the epidemiological, genetic, and environmental factors driving this cancer.

There is an urgent need to develop new targeted treatment agents for small cell lung cancer (SCLC). Tinengotinib (TT‐00420) is a novel, multi‐targeted, and spectrally selective small‐molecule kinase inhibitor that has shown significant inhibitory effects on certain solid tumors in preclinical studies. However, its role and mechanism of action in SCLC remain unclear. In this study, we demonstrated that tinengotinib effectively inhibited SCLC cell proliferation, especially highly expressing NeuroD1 (SCLC‐N), in the SCLC cell line‐derived xenograft (CDX) model and the malignant pleural effusion cell model of patients with SCLC. When combined with etoposide/cisplatin, it synergistically inhibited SCLC growth. Tinengotinib regulates proliferation, apoptosis, migration, cell cycle and angiogenesis in SCLC cells. Mechanistic studies revealed that c‐Myc expression may be a key factor influencing the effect of tinengotinib in SCLC‐N. This study provides reliable preclinical data and a new direction for tinengotinib as a promising therapy for SCLC, either alone or in combination with chemotherapy.

Lymph node metastasis significantly affects the NSCLC patients' staging, treatment strategy, and prognosis. Studies have shown that IGF2BP3, an oncofetal protein and an m6A reader, overexpresses and correlates to lymph node metastasis and worse overall survival in histopathological studies including NSCLC, but its mechanism needs further study. This study explored IGF2BP3's function and mechanism in LUAD lymphatic metastasis using public databases, a human LUAD tissue microarray, human LUAD cells, and a lymphatic metastasis model in male BALB/c nude mice. Firstly, we proved that IGF2BP3 overexpression was positively correlated to patients' lymph node metastasis and worse overall survival in bioinformatics and a human LUAD tissue microarray analysis. IGF2BP3 was knocked out or overexpressed in human LUAD cell lines. Functionally, IGF2BP3 facilitated NCI‐H1299, NCI‐H358, and A549 cell growth, migration, invasion, and EMT in vitro, and promoted tumorigenesis, lymphangiogenesis, and lymphatic metastasis of NCI‐H1299 cells in BALB/c nude mice. Mechanically, RIP, RNA pull‐down assay, MeRIP, mRNA stability assays, rescue experiments, and immunohistochemical assays were conducted. IGF2BP3 was demonstrated to bind to the m6A site of the 3′UTR region of SRC, stabilizing its mRNA and activating the downstream STAT3 signaling pathway and lymphatic growth factors such as VEGF‐C, therefore affecting lymphatic metastasis. The cell migration and EMT function of IGF2BP3 were partially rescued by utilizing SRC siRNA or AZD0530, an SRC inhibitor. This study demonstrated that IGF2BP3 promotes lymphatic metastasis in LUAD via activating the m6A‐SRC‐STAT3‐VEGFC signaling axis, indicating that IGF2BP3 is a potential therapeutic target to overcome metastasis in LUAD patients.

This study analyzed targeted sequencing data from 6530 tissue samples from patients with metastatic Chinese colorectal cancer (CRC) to identify low mutation frequency and subgroup‐specific driver genes, using three algorithms for overall CRC as well as across different clinicopathological subgroups. We analyzed 425 cancer‐related genes, identifying 101 potential driver genes, including 36 novel to CRC. Notably, some genes demonstrated subgroup specificity; for instance, ERBB4 was found as a male‐specific driver gene and mutations of ERBB4 only influenced the prognosis of male patients with CRC. This sex disparity of ERBB4 was validated in an independent large‐scale Memorial Sloan Kettering Cancer Center CRC cohort with 2444 samples. Furthermore, using network‐based stratification based on protein–protein interaction, we classified the microsatellite stable (MSS) and unstable (MSI) CRCs into six and three major subtypes, respectively, each showing unique phenotypes and prognoses. In MSS CRC, cluster 5 (APCAMER1–KRAS) and cluster 2 (RNF43–BRAF–PIK3CA) were predominant, and cluster 5 showed a superior overall survival compared with cluster 2. This extensive heterogeneity in driver gene mutations underscores the complexity of CRC and suggests significant implications for treatment and prognostic assessments.

Immunotherapy has revolutionized cancer treatment, making it a challenge to noninvasively monitor immune infiltration. Metabolic reprogramming in cancers, including hepatocellular carcinoma (HCC), is closely linked to immune status. In this study, we aimed to evaluate the ability of carbon‐11 acetate (¹¹C‐acetate) and fluorine‐18 fluorodeoxyglucose (¹⁸F‐FDG) PET/CT findings in predicting overall survival (OS) and immune infiltration in HCC patients. Totally 32 patients who underwent preoperative ¹⁸F‐FDG and ¹¹C‐acetate PET/CT, followed by liver resection for HCC, were prospectively enrolled at authors' institute between January 2019 and October 2021. Tracer uptake was qualified. Densities of CD3⁺, CD8⁺, and granzyme B⁺ CD8⁺ immune cells were assessed and the Immunoscore was defined by combining the densities of CD3⁺ and CD8⁺ in tumor interior (TI) and invasion margin (IM). Patients with avid HCCs in ¹¹C‐acetate PET/CT demonstrated a longer OS. Those with only ¹¹C‐acetate‐avid HCCs exhibited a longer OS compared to those with only ¹⁸F‐FDG uptake. In contrast to ¹⁸F‐FDG uptake, ¹¹C‐acetate uptake was positively associated with CD3⁺, CD8⁺, and granzyme B⁺ CD8⁺ cell infiltration. Patients with a higher Immunoscore exhibited a longer OS and an increased uptake of ¹¹C‐acetate rather than ¹⁸F‐FDG. The sensitivity of ¹¹C‐acetate PET/CT in the detection of patients with immune infiltration was superior to that of ¹⁸F‐FDG PET/CT (88% [21 of 24] vs. 58% [14 of 24]). These data show that preoperative ¹¹C‐acetate PET/CT may be a promising approach for the evaluation of immune status and postoperative outcome of HCCs.

Invasion and metastasis are major causes of mortality in breast cancer (BRCA) patients. LHPP, known for its tumor‐suppressive effects, has an undefined role in BRCA. We found reduced LHPP protein in BRCA tissues, with lower levels correlating with poor patient outcomes. In vitro studies show LHPP inhibits BRCA cell proliferation, migration, invasion, and stemness. In vivo xenograft models support LHPP's role in curbing tumorigenesis and lung metastasis. Mechanistically, LHPP interacts with ERK and P38 MAPK, leading to their dephosphorylation and suppression of the MAPK pathway. We also reveal ETS1, a MAPK effector, repressing LHPP mRNA transcription, suggesting a LHPP‐P38 MAPK/ERK‐ETS1 negative feedback loop as a key regulatory mechanism in controlling BRCA invasion and metastasis.

Effective therapeutic strategies for epithelioid sarcoma (EpS), a high‐grade soft tissue sarcoma characterized by loss of integrase interactor 1 (INI1), have not yet been developed. The present study therefore investigated the association between INI1 loss and upregulation of the aurora kinase A (AURKA)/polo‐like kinase 1 (PLK1)/cell division cycle 25C (CDC25C) axis, as well as the therapeutic relevance of this axis in EpS. Notably, our findings showed that the reintroduction of INI1 in VA‐ES‐BJ cells significantly reduced proliferation, mitigated tumorigenicity, and negatively regulated the expression of AURKA and its downstream effectors, as well as the activation of PLK1 and CDC25C. These results suggest that INI1 deficiency enhanced EpS growth by upregulating the AURKA/PLK1/CDC25C axis. AURKA silencing using siRNAs inhibited VA‐ES‐BJ and Asra‐EPS cell proliferation by inactivating PLK1 and CDC25C. Alisertib, a selective AURKA inhibitor, exerted markedly greater antiproliferative effects on EpS cells than on normal human dermal fibroblasts, and these effects were dependent on INI1 deficiency. Inhibition of AURKA activity by alisertib induced G2/M cell cycle arrest and apoptosis via the inactivation of AURKA downstream effectors in EpS cells. Alisertib also significantly decreased VA‐ES‐BJ xenograft tumor growth. Taken together, our findings revealed that INI1 loss in EpS cells enhances the expression of AURKA and its downstream effectors and persistently activates PLK1 and CDC25C mediated by AURKA, making the cells reliant on the AURKA/PLK1/CDC25C axis. Therefore, the AURKA/PLK1/CDC25C axis activated by INI1 deficiency could serve as a novel therapeutic target for this devastating disease.

The active vitamin D‐degrading enzyme (CYP24A1) is commonly overexpressed in various types of cancer, which is associated with poor prognosis in cancer patients. Recent studies highlight the antagonism of CYP24A1 toward the anticancer role of active vitamin D. However, the impact of CYP24A1 on tumorigenesis and its underlying mechanisms largely remains unexplored. This study also found that high CYP24A1 mRNA expressions were associated with poor prognosis in ovarian cancer and lung adenocarcinoma (LUAD) patients. Moreover, we demonstrated that the overexpression of CYP24A1 accelerated the proliferation, migration, and invasion of ovarian cancer and LUAD cancer cells in vitro. Furthermore, knockdown of CYP24A1 displayed an anticancer effector both in vitro and in vivo. Mechanically, 87–297 amino acid motif of CYP24A1 bound specifically to FUS protein, consequentially reducing FUS affinity for miR‐200c. Considering FUS promotes gene silencing by binding to microRNA targets, a decrease in miR‐200c levels led to a notable activation of its target ZEB1, resulting in the promotion of the epithelial‐mesenchymal transition (EMT) process. In conclusion, FUS binding specifically by CYP24A1 impaired miR‐200c‐mediated ZEB1 silencing, thereby augmenting EMT progression and tumorigenesis. These findings elucidate a fundamental mechanism by which CYP24A1 operates as an oncogene, offering potential targets for therapeutic interventions in cancer treatment.

Endometrial cancer (EC) is a worldwide gynecologic malignancies, with a remarking increase of incidence and mortality rates in recent years. Growing evidence indicates that glucose metabolism reprogramming is the most representative metabolic signature of tumor cells and exploring its modulatory function in EC development will promote identifying potential EC therapeutic targets. IGFBP2 is an insulin‐like growth factor binding protein which is closely associated with a variety of metabolic diseases. However, its biological role in EC and its effects on glucose metabolism remain unclear. In this study, we demonstrated that IGFBP2 was highly expressed in EC tissues and correlated with poor prognosis. Overexpression of IGFBP2 promoted proliferation and glycolysis in EC cells, whereas IGFBP2 knockdown had the opposite effect. Mechanistically, IGFBP2 directly interacted with PKM2, inducing weakened PKM2 protein degradation, and knockdown IGFBP2 expression prevented the translocation of PKM2 to the nucleus. Additionally, IGFBP2 expression was upregulated under the condition of hypoxia which directly regulated by transcriptional activation of HIF‐1α. Finally, the role of the IGFBP2/PKM2/HIF‐1α axis in EC tumor growth was confirmed in vivo using mouse xenograft models. Taken together, the current study identifies IGFBP2 as an upstream activator of PKM2‐driven proliferation and glycolysis in EC cells, providing a promising therapeutic target for EC.

In Japan, comprehensive genome profiling (CGP) as a companion diagnostic (CDx) has been covered by public insurance since June 2019, but the proportion of patients with cancer who actually received drug therapy based on CGP data is low. In the present study, we attempted to use CGP as a starting point for tumor‐informed circulating tumor DNA (ctDNA) monitoring. We retrospectively validated 219 patients with malignant tumors who underwent CGP at Iwate Medical University Hospital between October 2019 and April 2023 in terms of patient demographics, genetic analysis, drug recommendations, and drug administration rate. The 219 cancer cases analyzed by CGP for 27 target organs, including prostate (n = 27, 12.3%), colorectal (n = 25, 11.4%), lung (n = 19, 8.7%), and other neoplasms (n = 148, 67.6%). Among the cohort, only 14 cases (6.4%) subsequently were able to undertake the recommended action by Molecular Tumor Board. Of patients who underwent ctDNA monitoring based on somatic mutations identified by CGP (n = 11), clinical validity was confirmed in terms of early relapse prediction (n = 5, 45.5%), treatment response evaluation (n = 10, 90.9%), and no relapse/regrowth corroboration (n = 2, 18.2%) whereas 90.9% (n = 10) of patients obtained information with at least one source of the clinical validity. Although the current rate of CGP contributing to a drug recommendation is low, CGP results can be an alternate resource for tumor‐informed longitudinal ctDNA monitoring to provide information concerning early relapse prediction, treatment response evaluation, and no relapse/regrowth corroboration.

Small extracellular vesicles (sEVs) facilitate intercellular communication and play a pivotal role in tumor progression. Accumulated evidence has indicated the diversity of sEVs but with limited results revealing the landscape of heterogeneity of sEVs. The heterogeneity of cargo RNA in sEVs presents the different cell origins and indicates different functions. Here, we analyzed the heterogeneity of sEVs at droplet levels from single‐cell RNA sequencing results of head and neck squamous cell carcinoma (HNSCC) with the previously reported algorithm SEVtras. With the sEVs secretion activity calculated by SEVtras, we also found that the T cells held the major role of sEVs secretion. In addition, we found these sEVs secreted by T cells increased the cytotoxic ability of natural killer cells (NK cells), which illustrated an indirect manner for the anti‐tumor function of T cells. These results revealed the heterogeneity of cargo RNA of sEVs in HNSCC and underlined a sEVs‐dependent manner in which T cells act on NK cells and anti‐tumor immunity.

Cancer‐associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME). Given their various roles in tumor progression and treatment resistance, CAFs are promising therapeutic targets in cancer. The elimination of tumor‐promoting CAFs has been investigated in various animal models to determine whether it effectively suppresses tumor growth. Based on recent evidence, several simple strategies have been proposed to eliminate tumor‐promoting CAFs and attenuate these features. In addition, attention has focused on the critical role that CAFs play in the immunosuppressive TME. Therefore, the functional reprogramming of CAFs in combination with immune checkpoint inhibitors has also been investigated as a possible therapeutic approach. However, although potential targets in CAFs have been widely characterized, the plasticity and heterogeneity of CAFs complicate the understanding of their properties and present difficulties for clinical application. Moreover, the identification of tumor‐suppressive CAFs highlights the necessity for the development of therapeutic approaches that can distinguish and switch between tumor‐promoting and tumor‐suppressive CAFs in an appropriate manner. In this review, we introduce the origins and diversity of CAFs, their role in cancer, and current therapeutic strategies aimed at targeting CAFs, including ongoing clinical evaluations.

Third‐generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) is the standard therapy for patients harboring T790M after first‐generation EGFR‐TKI resistance. However, the impact of acquired EGFR amplification on the efficacy of third‐generation EGFR‐TKI against T790M remains uncertain. We aimed to investigate whether the presence of acquired EGFR amplification after first‐generation EGFR‐TKI resistance influences the efficacy of third‐generation EGFR‐TKI in patients with advanced non‐small‐cell lung cancer (NSCLC). We reviewed data from 275 advanced NSCLC patients harboring T790M after first‐generation EGFR‐TKI resistance. Patients were categorized into two groups based on the presence or absence of acquired EGFR amplification identified through next‐generation sequencing (NGS) after first‐line EGFR‐TKI treatment. We evaluated the efficacy of osimertinib used as a second‐line treatment. Among these patients, 59 exhibited acquired EGFR amplification, while 216 did not. The median progression‐free survival (PFS) was 12.20 months in the EGFR amplification group and 12.03 months in the non‐amplification group (p = 0.011), with median overall survival (OS) of 33.90 months and 23.30 months, respectively (p = 0.164). Multivariate analysis of PFS revealed that acquired EGFR amplification and EGFR 19del were independent prognostic factors for patients with T790M undergoing osimertinib. Additionally, subgroup analysis indicated a prolonged PFS in patients with EGFR 19del compared to those with EGFR 21L858R (p = 0.034) in the EGFR amplification group. Following first‐generation EGFR‐TKI resistance, advanced EGFR‐mutant NSCLC patients harboring both acquired T790M and EGFR amplification are likely to experience enhanced PFS with osimertinib. This phenomenon is particularly noteworthy among individuals with EGFR 19del.

Patient‐derived organoids represent a novel platform to recapitulate the cancer cells in the patient tissue. While cancer heterogeneity has been extensively studied by a number of omics approaches, little is known about the spatiotemporal kinase activity dynamics. Here we applied a live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients to comprehensively understand their heterogeneous growth potential and drug responses. By automated wide‐area image acquisitions and analyses, the PDAC cells were non‐selectively observed to evaluate their heterogeneous growth patterns. We monitored single‐cell ERK and AMPK activities to relate cellular dynamics to molecular dynamics. Furthermore, we evaluated two anti‐cancer drugs, a MEK inhibitor, PD0325901, and an autophagy inhibitor, hydroxychloroquine (HCQ), by our analysis platform. Our analyses revealed a phase‐dependent regulation of PDAC organoid growth, where ERK activity is necessary for the early phase and AMPK activity is necessary for the late stage of organoid growth. Consistently, we found PD0325901 and HCQ target distinct organoid populations, revealing their combination is widely effective to the heterogeneous cancer cell population in a range of PDAC patient‐derived organoid lines. Together, our live imaging quantitatively characterized the growth and drug sensitivity of human PDAC organoids at multiple levels: in single cells, single organoids, and individual patients. This study will pave the way for understanding the cancer heterogeneity and promote the development of new drugs that eradicate intractable cancer.

KRAS was long deemed undruggable until the discovery of the switch‐II pocket facilitated the development of specific KRAS inhibitors. Despite their introduction into clinical practice, resistance mechanisms can limit their effectiveness. Initially, tumors rely on mutant KRAS, but as they progress, they may shift to alternative pathways, resulting in intrinsic resistance. This resistance can stem from mechanisms like epithelial‐to‐mesenchymal transition (EMT), YAP activation, or KEAP1 mutations. KRAS inhibition often triggers cellular rewiring to counteract therapeutic pressure. For instance, feedback reactivation of signaling pathways such as MAPK, mediated by receptor tyrosine kinases, supports tumor cell survival. Inhibiting KRAS disrupts protein homeostasis, but reactivation of MAPK or AKT can restore it, aiding tumor cell survival. KRAS inhibition also causes metabolic reprogramming and protein re‐localization. The re‐localization of E‐cadherin and Scribble from the membrane to the cytosol causes YAP to translocate to the nucleus, where it drives MRAS transcription, leading to MAPK reactivation. Emerging evidence indicates that changes in cell identity, such as mucinous differentiation, shifts from alveolar type 2 to type 1 cells, or lineage switching from adenocarcinoma to squamous cell carcinoma, also contribute to resistance. In addition to these nongenetic mechanisms, secondary mutations in KRAS or alterations in upstream/downstream signaling proteins can cause acquired resistance. Secondary mutations in the switch‐II pocket disrupt drug binding, and known oncogenic mutations affect drug efficacy. Overcoming these resistance mechanisms involves enhancing the efficacy of drugs targeting mutant KRAS, developing broad‐spectrum inhibitors, combining therapies targeting multiple pathways, and integrating immune checkpoint inhibitors.

In this study, we investigated the measurable residual leukemic stem cell (MR‐LSC) population after allogeneic stem cell transplantation (allo‐SCT) for high‐risk acute myeloid leukemia (AML), utilizing T‐cell immunoglobulin mucin‐3 (TIM‐3) expression as a functional marker of AML leukemic stem cells (LSCs). Analysis of the CD34⁺CD38⁻ fraction of bone marrow cells immediately after achievement of engraftment revealed the presence of both TIM‐3⁺LSCs and TIM‐3⁻ donor hematopoietic stem cells (HSCs) at varying ratios. Genetic analysis confirmed that TIM‐3⁺ cells harbored patient‐specific mutations identical to those found in AML clones, whereas TIM‐3⁻ cells did not, indicating that TIM‐3⁺CD34⁺CD38⁻ cells represent residual AML LSCs. In 92 allo‐SCT occasions involving 83 AML patients, we enumerated the frequencies of TIM‐3⁺LSCs immediately after achieving hematologic complete remission with complete donor cell chimerism. Notably, only 22.2% of patients who achieved a TIM‐3⁺MR‐LSClow status (<60%) experienced relapse, with a median event‐free survival (EFS) of 1581 days (median follow‐up duration was 2177 days among event‐free survivors). Conversely, 87.5% of patients with TIM‐3⁺MR‐LSCint/high (≥60%) relapsed, with a median EFS of 140.5 days. Furthermore, MR‐LSC status emerged as a significant independent risk factor for relapse (hazard ratio, 8.56; p < 0.0001), surpassing the impact of patient disease status prior to allo‐SCT, including failure to achieve complete remission (hazard ratio, 1.98; p = 0.048). These findings suggest that evaluating TIM‐3⁺ MR‐LSCs immediately after engraftment, which reflects the competitive reconstitution of residual TIM‐3⁺ LSCs and donor HSCs, may be valuable for predicting outcomes in AML patients undergoing allo‐SCT.

Urothelial carcinoma (UC) can arise from either the lower urinary tract or the upper tract; they represent different disease entities and require different clinical treatment strategies. A full understanding of the cellular characteristics in UC may guide the development of novel therapies. Here, we performed single‐cell transcriptome analysis from four patients with UC of the bladder (UCB), five patients with UC of the ureter (UCU), and four patients with UC of the renal pelvis (UCRP) to develop a comprehensive cell atlas of UC. We found the rare epithelial cell subtype EP9 with epithelial‐to‐mesenchymal transition (EMT) and cancer stem cell (CSC) features, and specifically expressed SOX6, which was associated with poor prognosis. We also found that ACKR1+ endothelial cells and inflammatory cancer‐associated fibroblasts (iCAFs) were more enriched in UCU, which may promote pathogenesis. While ESM1+ endothelial cells may more actively participate in UCB and UCRP tumorigenesis by promoting angiogenesis. Additionally, CD8 + effector T cells were more enriched in UCU and UCRP patients, while Tregs were mainly enriched in UCB tumors. C1QC+ macrophages and LAMP3+ dendritic cells were more enriched in UCB, which is closely related to the formation of the heterogeneous immunosuppressive microenvironment. Furthermore, we found strong interactions between iCAFs, EP9, and Endo_ESM1, and different degrees of activation of the FGF‐FGFR3 axis and immune checkpoint pathway were observed in different UC subtypes. Our study elucidated the cellular heterogeneity and the components of the microenvironment in UC arising from the upper and lower urinary tracts and provided novel therapeutic targets.

Ovarian carcinosarcoma (OCS) is a rare and aggressive tumor, and the development of its sarcomatous component is believed to be due to epithelial–mesenchymal transition (EMT). The SWIch/sucrose nonfermentable chromatin remodeling factor (CRF) is closely related to EMT; however, the relationship between CRF and EMT in OCS remains unclear. In this study, we analyzed the protein expression of CRFs, including ARID1A and SMARCA4, and their downstream mRNA expression in 28 OCS cases, two fallopian tube CS cases, and one peritoneal CS case. ARID1A and SMARCA4 exhibited a histological type‐specific loss of protein expression in 5 of 11 (45%) endometrioid cases and all 5 serous/homologous OCS cases, respectively. The mRNA analysis suggested that sarcomatogenesis is induced by the transforming growth factor‐β and Hippo signaling pathways, both of which regulate YAP1. Immunostaining for YAP1 suggested YAP1‐associated sarcomatogenesis in the CRF‐retained group, whereas YAP1‐unassociated sarcomatogenesis was suggested in the CRF‐reduced group. High‐grade serous carcinoma cell line experiments showed that the transcriptome of the SMARCA4‐knockdown group showed lower expression of the epithelial gene CDH1 and higher expression of mesenchymal genes such as VIM, ZEB1, and SNAI1 than the control group. Moreover, cell adhesion disappeared and cell morphology changed to a spindle shape, indicating sarcomatogenesis. In conclusion, this study reveals a mechanism for sarcoma development in OCS and provides novel therapeutic possibilities.

Melanoma‐associated antigen (MAGE)‐A4, a cancer testis antigen, presents a promising target for chimeric antigen receptor T cell therapy in refractory solid tumors, including breast cancer (BC). However, the lack of highly specific Abs against MAGE‐A4 is a major challenge for the development of MAGE‐A4‐targeted immunotherapies. This study aimed to validate the specificity of a novel MAGE‐A4 Ab (E701U) and examine MAGE‐A4 expression in clinical BC samples. MAGE‐A1, ‐A2B, ‐A3, ‐A4, ‐A6, ‐A9, ‐A10, and ‐A12 genes were transfected into HEK293 cells. MAGE‐A4 expression in each inserted cell block was evaluated using an E701U Ab. Subsequently, we evaluated MAGE‐A4 expression in 403 primary BC tissue samples by immunohistochemistry using E701U and analyzed the clinical impact of MAGE‐A4 in patients with early BC. The results showed that MAGE‐A4 expression was limited to cells transduced with the MAGE‐A4 gene. MAGE‐A4 expression was observed in 5.7% of the BC samples. Positivity in triple‐negative BC was significantly higher than in the other subtypes. The 5‐year overall survival rate of patients with MAGE‐A4(+) was significantly worse than those with MAGE‐A4(−) BC. Moreover, the 5‐year recurrence‐free survival (RFS) rate of patients with MAGE‐A4(+) BC was significantly lower than that of patients with MAGE‐A4(−) BC. MAGE‐A4 expression was an independent prognostic factor for RFS. In conclusion, the E701U Ab showed reliable specificity for MAGE‐A4 expression among MAGE family genes. Patients with MAGE‐A4(+) BC have an unfavorable prognosis and represent potential candidates for MAGE‐A4‐specific immunotherapy.