Unveiling the intra-tumor fate of trastuzumab deruxtecan in a xenograft model to support its mechanism of action.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate used for cancer treatment comprising an anti-human epidermal growth factor receptor type 2 (HER2) antibody and the topoisomerase I inhibitor DXd. The present study investigated the intratumor fate of T-DXd. Fluorescence-labeled T-DXd was found to accumulate in tumors of HER2-positive tumor xenograft mice and was observed to be distributed within lysosomes of in vitro tumor cells in accordance with their HER2 expression. DXd was released by cysteine proteases, including cathepsins, in lysosomal fractions in vitro in response to the pH. Tumor slices obtained from HER2-positive tumor xenograft mice treated with T-DXd were examined by semi-quantitative and three-dimensional immunohistochemical assays using phosphor-integrated dots, which visualized DXd-related signals in the nucleus, the site of topoisomerase I inhibition. In addition, based on the data showing the antibody component of T-DXd barely distributed in the nucleus, it was suggested that the DXd-related signals detected in the nucleus were predominantly derived from free DXd. These observations help support the mode of action of T-DXd from the perspective of drug disposition.

Immuno-genomic analysis reveals eosinophilic feature and favorable prognosis of female non-smoking esophageal squamous cell carcinomas.

Most of esophageal squamous cell carcinoma (ESCC) develop in smoking males in Japan, but the genomic etiology and immunological characteristics of rare non-smoking female ECSS remain unclear. To elucidate the genomic and immunological features of ESCC in non-smoking females, we analyzed whole-genome or transcriptome sequencing data from 94 ESCCs, including 20 rare non-smoking female cases. In addition, 31,611 immune cells were extracted from four ESCC tissues and subject to single-cell RNA-seq. We compared their immuno-genomic and microbiome profiles between non-smoking female and smoking ESCCs. Non-smoking females showed much better prognosis. Whole-genome sequencing analysis showed no significant differences in driver genes or copy number alterations depending on smoking status. The mutational signatures specifically observed in non-smoking females ESCC could be attributed to aging. Immune profiling from RNA-seq revealed that ESCC in non-smoking females had high tumor microenvironment signatures and a high abundance of eosinophils with a favorable prognosis. Single-cell RNA-sequencing of intratumor immune cells revealed gender differences of eosinophils and their activation in female cases. ESCCs in non-smoking females have age-related mutational signatures and gender-specific tumor immune environment with eosinophils, which is likely to contribute to their favorable prognosis.

Pulmonary inflammatory leiomyosarcoma represents a potential diagnostic pitfall of DNA methylation-based classification of sarcomas: a case report.

BACKGROUND: Pulmonary inflammatory leiomyosarcoma (PILMS) is a rare type of myogenic tumor with prominent lymphohistiocytic infiltration. Despite their histological similarities, PILMS is immunohistochemically and genetically distinct from soft tissue inflammatory leiomyosarcoma, and its clinicopathological picture including DNA methylome data remains still unknown. CASE PRESENTATION: Here we present a case of PILMS in an 18-year-old male who underwent lobectomy. As reported previously, the current case demonstrated spindle myoid cell proliferation with smooth muscle differentiation within a prominent lymphohistiocytic infiltration and a diploid genome with a MUC3A gene alteration. DNA methylation analysis predicted this case to be an "inflammatory myofibroblastic tumor" (IMT) according to the Deutsches Krebsforschungszentrum (DKFZ) classifier. The data was analyzed by t-distributed stochastic neighbor embedding, which plotted the case tumor in the vicinity of IMT, however, there were no IMT histological features. These discordant results could be due to background non-neoplastic inflammatory cells. CONCLUSIONS: As the DNA methylation classification of PILMS might be a potential diagnostic pitfall, an integrative histological and genetic approach is required for its accurate diagnosis.

Physiologically Based Pharmacokinetic Modeling for Quantitative Prediction of Exposure to a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Substrate of Cytochrome P450 and Aldehyde Oxidase, Using Chimeric Mice With Humanized Liver.

In a previous study on the human mass balance of DS-1971a, a selective NaV1.7 inhibitor, its CYP2C8-dependent metabolite M1 was identified as a human disproportionate metabolite. The present study assessed the usefulness of pharmacokinetic evaluation in chimeric mice grafted with human hepatocytes (PXB-mice) and physiologically based pharmacokinetic (PBPK) simulation of M1. After oral administration of radiolabeled DS-1971a, the most abundant metabolite in the plasma, urine, and feces of PXB-mice was M1, while those of control SCID mice were aldehyde oxidase-related metabolites including M4, suggesting a drastic difference in the metabolism between these mouse strains. From a qualitative perspective, the metabolite profile observed in PXB-mice was remarkably similar to that in humans, but the quantitative evaluation indicated that the area under the plasma concentration-time curve (AUC) ratio of M1 to DS-1971a (M1/P ratio) was approximately only half of that in humans. A PXB-mouse-derived PBPK model was then constructed to achieve a more accurate prediction, giving an M1/P ratio (1.3) closer to that in humans (1.6) than the observed value in PXB-mice (0.69). In addition, simulated maximum plasma concentration and AUC values of M1 (3429 ng/ml and 17,116 ng·h/ml, respectively) were similar to those in humans (3180 ng/ml and 18,400 ng·h/ml, respectively). These results suggest that PBPK modeling incorporating pharmacokinetic parameters obtained with PXB-mice is useful for quantitatively predicting exposure to human disproportionate metabolites. SIGNIFICANCE STATEMENT: The quantitative prediction of human disproportionate metabolites remains challenging. This paper reports on a successful case study on the practical estimation of exposure (C max and AUC) to DS-1971a and its CYP2C8-dependent, human disproportionate metabolite M1, by PBPK simulation utilizing pharmacokinetic parameters obtained from PXB-mice and in vitro kinetics in human liver fractions. This work adds to the growing knowledge regarding metabolite exposure estimation by static and dynamic models.

Estimation of fraction of drug metabolism by a single UDP-glucuronosyl transferase enzyme using relative expression factor.

The estimation of the contributions of UDP-glucuronosyl transferase (UGT) isoforms to the overall metabolism still suffers from technical difficulties due to limited information on enzyme levels in recombinant systems and specific inhibitors, unlike the case for cytochrome P450s (CYPs). The protein expression levels of UGT in both recombinant system microsomes (RM) and human liver microsomes (HLM) were quantified using liquid chromatography-tandem mass spectrometry, and the relative expression factor (REF) value of HLM to recombinant microsomes was estimated to evaluate the fractions of drug metabolism by a single UGT enzyme (fmUGT) of UGT substrates. The REF values of UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17 were 0.228, 0.0714, 0.0665, 0.420, 0.118, and 0.0442, respectively. fmUGTs in HLM were estimated for several typical UGT substrates utilizing these values and metabolic clearances in RM. These values were comparable to the reported values estimated by various methods. This study provided useful information on REF values, which promote a robust estimation of fmUGT values for UGT substrates when evaluating the contribution of UGT isoforms to total metabolic clearance.

Cytokeratin-positive Malignant Tumor in the Abdomen With EWSR1/FUS-CREB Fusion: A Clinicopathologic Study of 8 Cases.

ATF1, CREB1, and CREM, which encode the CREB family of transcription factors, are fused with EWSR1 or FUS in human neoplasms, such as angiomatoid fibrous histiocytoma. EWSR1/FUS-CREB fusions have recently been reported in a group of malignant epithelioid tumors with a predilection to the peritoneal cavity and frequent cytokeratin expression. Here, we studied 8 cytokeratin-positive abdominal malignancies with these fusions for further characterization. The tumors affected males (15 to 76 y old) and presented as intra-abdominal masses with concurrent or subsequent peritoneal dissemination, ascites, and/or metastases to the liver or lymph nodes. Four patients died of the disease within 18 to 140 months. Cases 1 to 5 showed multinodular growth of monomorphic epithelioid cells with focal serous cysts. Lymphoplasmacytic infiltration was prominent and was associated with systemic inflammatory symptoms. Two patients suffered from membranous nephropathy with nephrosis. The tumors displayed partly overlapping phenotypes with malignant mesothelioma, including diffuse strong expression of AE1/AE3 and WT1 and membranous positivity of sialylated HEG1, although calretinin was negative. Case 6 showed similar histology to cases 1 to 5, but expressed smooth muscle actin diffusely, lacked WT1 and HEG1, and harbored prominent pseudoangiomatous spaces. Cases 7 and 8 displayed dense growth of small oval to short spindle cells, with occasional molding and minor swirling, superficially resembling small cell carcinoma. Lymphoplasmacytic infiltration was not observed. The tumors were positive for AE1/AE3 and CD34 (focal), whereas calretinin, WT1, and HEG1 were negative. The detected fusions were FUS-CREM (n=4), EWSR1-ATF1 (n=2), EWSR1-CREB1 (n=1), and EWSR1-CREM (n=1). We confirmed the prior observation that these tumors do not fit perfectly with known entities and provided additional novel clinicopathologic information. The tumors require wider recognition because of more aggressive behavior than angiomatoid fibrous histiocytoma despite similar genetics, and potential misdiagnosis as unrelated diseases, such as neuroendocrine neoplasms.

CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate.

Predicting human disproportionate metabolites is difficult, especially when drugs undergo species-specific metabolism mediated by cytochrome P450s (P450s) and/or non-P450 enzymes. This study assessed human metabolites of DS-1971a, a potent Nav1.7-selective blocker, by performing human mass balance studies and characterizing DS-1971a metabolites, in accordance with the Metabolites in Safety Testing guidance. In addition, we investigated the mechanism by which the major human disproportionate metabolite (M1) was formed. After oral administration of radiolabeled DS-1971a, the major metabolites in human plasma were P450-mediated monoxidized metabolites M1 and M2 with area under the curve ratios of 27% and 10% of total drug-related exposure, respectively; the minor metabolites were dioxidized metabolites produced by aldehyde oxidase and P450s. By comparing exposure levels of M1 and M2 between humans and safety assessment animals, M1 but not M2 was found to be a human disproportionate metabolite, requiring further characterization under the Metabolites in Safety Testing guidance. Incubation studies with human liver microsomes indicated that CYP2C8 was responsible for the formation of M1. Docking simulation indicated that, in the formation of M1 and M2, there would be hydrogen bonding and/or electrostatic interactions between the pyrimidine and sulfonamide moieties of DS-1971a and amino acid residues Ser100, Ile102, Ile106, Thr107, and Asn217 in CYP2C8, and that the cyclohexane ring of DS-1971a would be located near the heme iron of CYP2C8. These results clearly indicate that M1 is the predominant metabolite in humans and a human disproportionate metabolite due to species-specific differences in metabolism. SIGNIFICANCE STATEMENT: This report is the first to show a human disproportionate metabolite generated by CYP2C8-mediated primary metabolism. We clearly demonstrate that DS-1971a, a mixed aldehyde oxidase and cytochrome P450 substrate, was predominantly metabolized by CYP2C8 to form M1, a human disproportionate metabolite. Species differences in the formation of M1 highlight the regio- and stereoselective metabolism by CYP2C8, and the proposed interaction between DS-1971a and CYP2C8 provides new knowledge of CYP2C8-mediated metabolism of cyclohexane-containing substrates.

Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase.

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Clinicopathologic Characterization of Epithelioid Hemangioendothelioma in a Series of 62 Cases: A Proposal of Risk Stratification and Identification of a Synaptophysin-positive Aggressive Subset.

Epithelioid hemangioendothelioma (EHE) is a rare vascular endothelial neoplasm with characteristic histology and distinctive fusion genes. Its clinical presentation and outcome are heterogeneous, and the determinants of survival are controversial. In this study, we aimed to identify clinicopathologic prognostic factors of EHE in a retrospective cohort of 62 cases with CAMTA1/TFE3/WWTR1 alterations. The tumors were of the CAMTA1 subtype for 59 cases, TFE3 subtype for 2 cases, and variant WWTR1 subtype (WWTR1-ACTL6A) for 1 case. Twenty-two tumors (35.5%) demonstrated atypical histology, defined by having at least 2 of the following 3 findings: high mitotic activity (>1/2 mm2), high nuclear grade, and coagulative necrosis. During a median follow-up of 34 months, 11 patients (18%) died, and the 5-year overall survival rate was 78.8%. Survival did not correlate with such clinical parameters as age, sex, tumor sites, multifocality, and multiorgan involvement. Conversely, based on both univariate and multivariate analyses, large tumor size (>30 mm) and histologic atypia were significantly associated with a shorter survival. A proposed 3-tiered risk assessment system using these 2 parameters significantly stratified the patients into low-risk, intermediate-risk, and high-risk groups with 5-year overall survival rates of 100%, 81.8%, and 16.9%, respectively (P<0.001). Four tumors (6.4%) expressed synaptophysin, which all belonged to the high-risk group and pursued an aggressive course. The present study demonstrated the independent prognostic significance of large tumor size and atypical histology in EHE, as well as the value of risk stratification using these 2 factors. Moreover, we revealed a small EHE subset with aberrant synaptophysin expression, which may have potential prognostic and diagnostic implications.

Two cases of breast angiosarcoma after breast conserving surgery.

BACKGROUND: Breast angiosarcoma (AS) is a rare malignant breast tumor arising from endothelial cells lining the blood vessels. The prognosis of AS is reportedly poor. However, the effectiveness of chemotherapy and radiation is still controversial. Surgery is the only curable treatment, and removal of AS with adequate surgical margin is important. CASE PRESENTATION: We report two cases of primary and radiation-induced breast angiosarcoma (AS) after performing breast conserving surgery (BCS) for breast cancer. In case 1, a 72-year-old woman underwent right BCS with adjuvant radiation therapy (RT) for breast cancer 5 years prior. She was diagnosed with AS of the right breast and underwent mastectomy with a wide skin resection of the breast. As the tumor cells were positive for c-myc, this tumor was diagnosed as a radiation-induced AS. In case 2, an 80-year-old woman underwent BCS without adjuvant RT. She was diagnosed with AS 3 years after BCS and underwent mastectomy with a wide skin resection of the breast. The tumor was diagnosed to be a primary AS because there were no episodes of RT or lymphedema. Both cases developed local recurrence within 1 year of surgery. CONCLUSION: Our cases suggest that surgical margin is associated with the risk of local recurrence, and the difficulty of deciding a safe surgical margin should be set during preoperative diagnosis.

Giant multilocular prostatic cystadenoma.

BACKGROUND: The giant multilocular prostatic cystadenoma is a very rare benign tumor of the prostate gland. It is composed of predominantly cystic enlarged prostatic glands in a fibrous stroma and spreads extensively into the pelvis. Because of the large size at the time of diagnosis, it is not always possible to determine the exact point of origin for these multilocular cystic neoplasms. Thus, diagnosis before histological examination of a surgical specimen is often difficult. Here, we present a case involving one of the largest giant multilocular prostatic cystadenomas reported in the literature and discuss preoperative diagnoses and appropriate surgical approaches for this rare retroperitoneal tumor. CASE PRESENTATION: A 50-year-old man presented with a 2-year history of abdominal distension and lower urinary symptoms. Enhanced CT showed a large retroperitoneal mass with multiple septations in the pelvis and lower abdomen, measuring 30 cm in size, surrounding the rectum and displacing the bladder, prostate, and seminal vesicle to the right anterior side. MRI showed multiple cysts with a simple fluid appearance on T2-weighted images and enhanced solid components on gadolinium-enhanced fat-saturated T1-weighted images, suggesting the retroperitoneal mass as leiomyoma with cystic degeneration or perivascular epithelioid cell tumor. Biopsy of the mass showed a spindle cell tumor with focal smooth muscle differentiation. Differential diagnosis comprising leiomyoma, low-grade leiomyosarcoma, and perivascular epithelioid cell tumor was made. Complete resection of the tumor with low anterior resection of the rectum was performed. The tumor was solid with multilocular cavities containing blackish-brown fluid and measured 33 × 23 × 10 cm. Histologically, the tumor was composed of variously sized dilated glandular structures lined by prostatic epithelia surrounded by fibromuscular stroma. The prostatic nature of the lesions was confirmed by immunohistochemical staining of the epithelium for prostate-specific antigen. Thus, pathological diagnosis was a giant multilocular prostatic cystadenoma. CONCLUSIONS: We present our experiences with one of the largest giant multilocular prostatic cystadenomas. When a retroperitoneal huge lesion with locular cavities fills the pelvis in a male patient, the possibility of giant multilocular prostatic cystadenoma should be considered before planning for retroperitoneal tumor treatment.

Challenging Differential Diagnosis of Hypergastremia and Hyperglucagonemia with Chronic Renal Failure: Report of a Case with Multiple Endocrine Neoplasia Type 1.

A 53-year-old woman developed end-stage renal failure during a 15-year clinical course of primary hyperparathyroidism and was referred to our hospital for evaluation of suspected multiple endocrine neoplasia type 1 (MEN1). Genetic testing revealed a novel deletion mutation at codon 467 in exon 10 of the MEN1 gene. Systemic and selective arterial calcium injection (SACI) testing revealed hyperglucagonemia and hypergastrinemia with positive gastrin responses. A pathological examination revealed glucagonoma and a lymph node gastrinoma. The findings in this case indicate the importance of early diagnosis of MEN1 and demonstrate the utility of systemic and SACI testing in renal failure cases.

[Metastatic Ovarian Granulosa Cell Tumor of the Diaphragm Resected by Video-assisted Thoracoscopic Surgery;Report of a Case].

A 75-years-old woman, who had undergone a total abdominal hysterectomy and bilateral salpingo-oophorectomy for ovarian granulosa cell tumor( OGCT) 6 years ago, was admitted to our hospital to treat a mass on the right diaphragm detected by computed tomography. The mass was resected successfully by video-assisted thoracoscopic surgery and pathological diagnosis of the tumor was a metastatic OGCT of the diaphragm. To our knowledge, this is the 3rd case report of metastatic OGCT of the diaphragm.

Loss of Hep Par 1 immunoreactivity in the livers of patients with carbamoyl phosphate synthetase 1 deficiency.

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D.

A novel selective prostaglandin E2 synthesis inhibitor relieves pyrexia and arthritis in Guinea pigs inflammatory models.

Prostaglandin E2 (PGE2), one of the terminal products in the cyclooxygenase pathway, plays an important role in various inflammatory responses. To determine whether selective inhibition of PGE2 may relieve these inflammatory symptoms, we synthesized a selective PGE2 synthesis inhibitor, compound A [1-(6-fluoro-5,7-dimethyl-1,3-benzothiazol-2-yl)-N-[(1S,2R)-2-(hydroxymethyl)cyclohexyl]piperidine-4-carboxamide], then investigated the effects on pyrexia, arthritis and inflammatory pain in guinea pigs. In LPS-stimulated guinea pig macrophages, compound A selectively inhibited inducible PGE2 biosynthesis in a dose-dependent manner whereas enhanced the formation of thromboxane B2 (TXB2). Compound A suppressed yeast-evoked PGE2 production selectively and enhanced the production of TXB2 and 6-keto PGF1αin vivo. In addition, compound A relieved yeast-induced pyrexia and also suppressed paw swelling in an adjuvant-induced arthritis model. The effect on gastrointestinal (GI) ulcer formation was also evaluated and compound A showed a lower GI adverse effect than indomethacin. However, compound A failed to relieve yeast-induced thermal hyperalgesia. These results suggest that selective inhibition of PGE2 synthesis may have anti-pyretic and anti-inflammatory properties without GI side effect, but lack the analgesic efficacy.

Screening of BCOR-CCNB3 sarcoma using immunohistochemistry for CCNB3: A clinicopathological report of three pediatric cases.

BCOR-CCNB3 sarcoma is a recently recognized tumor morphologically and clinically simulating Ewing sarcoma. We herein retrospectively collected clinicopathologic data on BCOR-CCNB3 sarcoma in the Kyoto University Hospital over the last 10 years. Three (20%) bone sarcomas were revealed to be diffusely positive for CCNB3 immunohistochemistry among 15 pediatric cases of undifferentiated sarcoma morphologically similar to Ewing sarcoma, while the other cases showed completely negative staining. The three patients with immunohistochemically CCNB3-positive tumors were all male, aged between 11 and 17, and confirmed to have the BCOR-CCNB3 fusion transcript by RT-PCR. Radiologically, all cases had well-demarcated solid masses with bone destruction. Although the tumors were basically small round cell tumors, less monomorphic histological patterns such as short spindle cells, a myxoid matrix, and hemangiopericytoma-like pattern were also observed in both biopsy and resected specimens. Two patients achieved a complete response after chemotherapy for Ewing sarcoma and osteosarcoma, respectively. These results demonstrated that the application of CCNB3 immunostaining was useful for differentiating BCOR-CCNB3 from a group of 'Ewing-like sarcomas' and may contribute to the evaluation of treatment strategies for bone sarcomas.