Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

BACKGROUND: Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. METHODS: We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. RESULTS: Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. CONCLUSIONS: Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

[A Case of the Huge GIST in the Transverse Colon Mesentery with Intra-Tumoral Hemorrhage and Jejunal Infiltration].

A 38-year-old woman was admitted to our hospital due to severe anemia. CT showed a 13×12 cm tumor with moderately enhanced wall thickening in the right upper abdomen. The huge tumor located adjacent to the jejunum and compressed the right transverse colon. Hemorrhagic necrosis and air were observed within the tumor, suspecting tumor penetration into the jejunum. The patient was diagnosed with abdominal GIST with jejunal infiltration. Laparotomy revealed a 13× 11 cm solid mass with intra-tumoral hemorrhage and invasion into the jejunum, located in the transverse mesocolon. Tumor resection combined with partial jejunectomy and transverse colectomy were performed. Immunohistochemical findings of the resected specimen was positive for c-kit and DOG-1, and the MIB-1 positive rate was 10%. Three weeks after the operation, re-anastomosis was performed due to transverse colon anastomotic stricture. She was discharged 45 days after first operation. Currently, 9 months after the operation, patient has been prescribed imatinib and is alive without recurrence.

Skin Manifestations of Micafungin Breakthrough Disseminated Trichosporonosis in Acute Megakaryoblastic Leukemia.

Disseminated trichosporonosis is a rare fungal infection whose risk factors are hematological malignancies and neutropenia. Recently, breakthrough Trichosporon infections after administration of micafungin, the first-line systemic antifungal agent in compromised hosts, have been widely recognized. A man in his seventies about 1 month into chemotherapy for acute megakaryoblastic leukemia presented with a worsening fever and dyspnea. The patient was being administered with empirical micafungin therapy for suspected candidiasis. As the symptoms progressed, scattered erythema appeared on the trunk, some with a dark red vesicle at the center. Blood cultures identified Trichosporon asahii, as did the specimen of the skin biopsy. On the basis also of the presence of pneumonia on chest computed tomography, we confirmed the diagnosis of disseminated trichosporonosis and changed the antifungal agent from micafungin to voriconazole. Blood culture turned out to be negative 1 month after administrating voriconazole. However, the patient died of the leukemia. Our review of previous reports on cutaneous manifestations of disseminated trichosporonosis revealed that despite their morphological diversity, erythema with a red papule or vesicle at the center, implying necrosis, was also observed in previous cases. Our case report suggests that dermatologists should be aware of skin manifestations of disseminated trichosporonosis after micafungin administration, especially in cases of hematological malignancies.

An ampullary adenoma presenting with jaundice caused by duodenal intussusception: a case report.

BACKGROUND: Ampullary adenomas are premalignant lesions. However, biliary obstruction causing jaundice is rare. Duodenal intussusception secondary to an ampullary adenoma rarely occurs because of the fixed position of the duodenum in the retroperitoneum. Herein, we have described a rare case of ampullary adenoma with jaundice caused by duodenal intussusception. CASE PRESENTATION: A 40-year-old woman presenting with vomiting and yellowish discoloration of the skin was admitted to another hospital. The patient had experienced recurrent epigastric pain and vomiting for the past 18 months. Blood test results showed elevated levels of bilirubin (3.9 mg/dL), and abdominal computed tomography (CT) showed a 60-mm hypovascular mass in the third part of the duodenum and a left lateral shift of the dilated common bile duct. The patient was referred to our hospital for further evaluation. She recovered from hyperbilirubinemia spontaneously (levels of bilirubin, 1.0 mg/dL), and the CT showed a tumor shift from the third part of the duodenum to the second part and improvement of the dilated common bile duct. Hypotonic duodenography revealed a tumor that moved easily from the second to the third portion of the patient's position. Upper gastrointestinal endoscopy revealed a large papillary tumor occupying the second part of the duodenum, which was diagnosed as an adenoma through biopsy. The possibility of malignancy could not be negated owing to the presence of jaundice and an elevated carbohydrate antigen 19-9 level (76.0 U/mL). Pancreaticoduodenectomy was performed. The resected specimen showed a 60 × 40 × 40-mm pedunculated ampullary mass with submucosal elongation. The pathological examination indicated that the ampullary tumor was a high-grade intestinal adenoma. The postoperative course was uneventful, and the patient was discharged 26 days postoperatively. CONCLUSIONS: This report describes a rare case of a patient with an ampullary adenoma presenting with jaundice resulting from duodenal intussusception. Owing to the possibility of a postoperative cancer diagnosis which may have caused the biliary obstruction and the difficulty in making an accurate preoperative diagnosis, it is imperative to choose the appropriate surgical procedure such as a pancreaticoduodenectomy.

Lymph node macrophages drive innate immune responses to enhance the anti-tumor efficacy of mRNA vaccines.

mRNA vaccines are promising for cancer treatment. Efficient delivery of mRNAs encoding tumor antigens to antigen-presenting cells (APCs) is critical to elicit anti-tumor immunity. Herein, we identified a novel lipid nanoparticle (LNP) formulation, L17-F05, for mRNA vaccines by screening 34 ionizable lipids and 28 LNP formulations using human primary APCs. Subcutaneous delivery of L17-F05 mRNA vaccine encoding Gp100 and Trp2 inhibited tumor growth and prolonged the survival of mice bearing B16F10 melanoma. L17-F05 efficiently delivered mRNAs to conventional dendritic cells (cDCs) and macrophages in draining lymph nodes (dLNs). cDCs functioned as the main APCs by presenting antigens along with enhanced expression of co-stimulatory molecules. Macrophages triggered innate immune responses centered on type-I interferon (IFN-I) in dLNs. Lymph node (LN) macrophage depletion attenuated APC maturation and anti-tumor activity of L17-F05 mRNA vaccines. Loss-of-function studies revealed that L17-F05 works as a self-adjuvant by activating the stimulator of interferon genes (STING) pathway in macrophages. Collectively, the self-adjuvanticity of L17-F05 triggered innate immune responses in LN macrophages via the STING-IFN-I pathway, contributing to APC maturation and potent anti-tumor activity of L17-F05 mRNA vaccines. Our findings provide strategies for further optimization of mRNA vaccines based on the innate immune response driven by LN macrophages.

Applicability of the FDA-approved Immunohistochemical Panel for Identification of MMRd Phenotype in Uterine Endometrioid Carcinoma.

Recently, the US Food and Drug Administration (FDA) approved the Ventana MMR RxDx Panel as the first immunohistochemical companion diagnostic test for identification of tumors with mismatch repair (MMR) status. The aim of this study was to investigate the accuracy of this test in comparison with polymerase chain reaction (PCR)-based microsatellite instability (MSI) analysis. We assessed the MMR/MSI concordance rate in 140 cases of endometrioid carcinoma. MMR status was evaluated by immunohistochemistry (MMR-IHC), and MSI status was evaluated by PCR-based analysis (MSI-PCR). Potential molecular mechanisms responsible for MSH6 staining variations were also analyzed. Immunohistochemistry showed that 34 tumors (24.3%) were MMRd; these included 26 with combined MLH1/PMS2 loss, 2 with combined MSH2/MSH6 loss, and 6 with isolated MSH6 loss. Heterogeneous MSH6 loss was found in 10 tumors and was recognized only in tumors with combined MLH1/PMS2 loss. Eight of 10 tumors with heterogeneous MSH6 loss harbored MSH6 C8 tract instability, suggesting a secondary somatic event after MLH1/PMS2 loss. MSI-PCR revealed that 102 tumors were MSS, 4 were MSI-low, and 34 were MSI-high. Consequently, MMR-IHC and MSI-PCR showed perfect concordance (kappa=0.080, P <0.0001). However, 10 of the 34 MSI-high tumors, including the 6 tumors with isolated MSH6 loss, showed only minimal microsatellite shift by MSI-PCR, which may have been erroneously interpreted as MSS or MSI-low. On the basis of these findings, we consider that the FDA-approved immunohistochemical panel can detect MMR variations consistently and is more accurate than MSI-PCR for determining the applicability of immune checkpoint inhibitors for treatment of endometrioid carcinomas.

Lipid nanoparticle-targeted mRNA formulation as a treatment for ornithine-transcarbamylase deficiency model mice.

Ornithine transcarbamylase (OTC) plays a significant role in the urea cycle, a metabolic pathway functioning in the liver to detoxify ammonia. OTC deficiency (OTCD) is the most prevalent urea cycle disorder. Here, we show that intravenously delivered human OTC (hOTC) mRNA by lipid nanoparticles (LNP) was an effective treatment for OTCD by restoring the urea cycle. We observed a homotrimer conformation of hOTC proteins produced by the mRNA-LNP in cells by cryo-electron microscopy. The immunohistochemistry revealed the mitochondria localization of produced hOTC proteins in hepatocytes in mice. In livers of mice intravenously injected with hOTC-mRNA/LNP at 1.0 mg/kg, the delivered hOTC mRNA levels steeply decreased with a half-life (t1/2) of 7.1 h, whereas the produced hOTC protein levels retained for 5 days and then declined with a t1/2 of 2.2 days. In OTCD model mice (high-protein diet-fed Otcspf-ash hemizygous males), a single dose of hOTC-mRNA/LNP at 3.0 mg/kg ameliorated hyperammonemia and weight loss with prolonged survival rate (22 days) compared with that of untreated mice (11 days). Weekly repeated doses at 0.3 and 1.0 mg/kg were well tolerated in wild-type mice and showed a dose-dependent amelioration of survival rate in OTCD mice, thus, showing the therapeutic potential of LNP-formulated hOTC mRNA for OTCD.

[A Patient with AFP-Producing Gastric Cancer and Stenosis Who Regained Oral Intake Capabilities after T-DXd Treatment].

We present the case of a 55-year-old man with HER2-positive, AFP-producing gastric cancer and multiple liver metastases. The patient consequently underwent 7 courses of SOX plus trastuzumab therapy, 3 courses of weekly PTX plus ramucirumab therapy, and 3 courses of nivolumab therapy, all of which resulted in PD. Obstruction due to tumor growth became noticeable 9 months after the start of the first treatment. Subsequently, the patient experienced malnutrition and systemic edema due to impaired oral intake. However, subsequent trastuzumab deruxtecan(T-DXd)therapy induced remarkable tumor shrinkage. Furthermore, oral intake became possible, and edema started subsiding. Thus, we report the course of a patient with AFP-producing gastric cancer and stenosis who regained oral intake capabilities after T-DXd treatment.

Prognostic value and clinicopathological roles of the tumor immune microenvironment in salivary duct carcinoma.

Salivary duct carcinoma (SDC) is an aggressive type of salivary gland carcinoma. Recently, immunotherapies targeting immune checkpoints, including PD1, PD-L1, CTLA4, and LAG3, have had a considerable prognostic impact on various malignant tumors. The implementation of such immune checkpoint inhibitor (ICI) therapies has also been attempted in cases of salivary gland carcinoma. The tumor immune microenvironment (TIME) is implicated in tumorigenesis and tumor progression and is closely associated with the response to ICI therapies. However, the TIME in SDC has not been fully explored. We examined the immunohistochemical expression of CD8, FOXP3, PD1, PD-L1, CTLA4, LAG3, and mismatch repair (MMR) proteins, tumor-infiltrating lymphocytes (TILs), and microsatellite instability (MSI) status in 175 cases of SDC. The associations between these TIME-related markers and the clinicopathological factors and prognosis were evaluated. An elevated expression of CD8, FOXP3, PD1, CTLA4, and LAG3 was associated with more aggressive histological features and an advanced N and/or M classification, elevated Ki-67 index, and poor prognosis. Furthermore, cases with a high PD-L1 expression exhibited more aggressive histological features and adverse clinical outcomes than those with a low expression. Alternatively, there was no significant correlation between TILs and clinicopathological factors. No SDC cases with an MSI-high status or MMR deficiency were found. The coexistence of both an immunostimulatory and immunosuppressive TIME in aggressive SDC might play a role in the presence of T-cell exhaustion. The contribution of multiple immune escape pathways, including regulatory T cells and immune checkpoints, may provide a rationale for ICI therapy, including combined PD1/CTLA4 blockade therapy.

Anti-glomerular Basement Membrane Disease Concomitant with MPO-ANCA Positivity Concurrent with High Serum Levels of Interleukin-26 Following Coronavirus Disease 2019 Vaccination.

As coronavirus disease 2019 (COVID-19) vaccine booster campaigns progress worldwide, new reports of complications following COVID-19 vaccination have emerged. We herein report a case of new-onset anti-glomerular basement membrane (GBM) disease concomitant with myeloperoxidase-antineutrophil cytoplasmic antibody positivity concurrent with high levels of interleukin (IL)-26 following the second dose of the Pfizer-BioNTech COVID-19 vaccine. The temporal association with vaccination in this case suggests that an enhanced neutrophilic immune response through IL-26 may have triggered necrotizing glomerulonephritis and a T-cell-mediated immune response to GBMs, leading to the development of anti-GBM antibodies, with an enhanced B-cell response after the vaccination triggering anti-GBM IgG and the onset of anti-GBM disease.

[A Case of Pancreatic Tail Cancer Causing Gastrointestinal Hemorrhage Due to Transverse Colon Invasion].

An 81-year-old woman was admitted to our hospital due to frequent bleeding and hemorrhagic shock. Blood tests revealed anemia and contrast-enhanced abdominal CT revealed a pancreatic tail tumor with a diameter of 60 mm. The boundary between pancreatic tumor and the transverse colon, stomach and spleen was unclear, and invasion of the transverse colon as well as the stomach and spleen was suspected. Hemorrhage due to colon invasion of the pancreatic tail cancer and intra-tumoral hemorrhage were suspected. Due to persistent bleeding, the patient had emergency surgery to control bleeding. The pancreatic tail tumor invaded not only the colon but also stomach and spleen, distal pancreatectomy, partial gastrectomy and splenectomy was performed in combination with resection of the transverse colon, and transverse colon colostomy. We report a case of gastrointestinal bleeding caused by transverse colon invasion of pancreatic tail cancer, which resulted in emergency surgery.

Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo.

Nucleic acid therapeutics have been utilized for gene regulation, and their recent advancement has led to approval of novel drugs for liver-related disorders. However, systemic extrahepatic delivery remains challenging. Here, we report newly designed mannose-conjugated oligonucleotides for delivering oligonucleotides to macrophages by leveraging the mannose receptor, C-type 1 (MRC1, CD206), which is abundantly expressed in macrophages. We investigated the relationship between cellular uptake and multivalency (mono to tetra) of mannose ligands or linker length and selected a trivalent-mannose ligand. Trivalent-mannose (Man3)-conjugated siRNA induced concentration-dependent gene silencing in both human CD206-overexpressing cells and human macrophages in vitro. After subcutaneous injection into mice, we observed a high distribution of Man3-conjugated oligonucleotides in the liver and pancreata as well as cellular uptake into Kupffer cells and pancreatic macrophages. A single subcutaneous injection of Man3-conjugated siRNA (10 mg/kg) targeting ß2-microglobulin (B2M) silenced B2m mRNA expression by ∼50% and decreased its protein levels in mouse pancreatic macrophages compared to those in saline-treated mice. Of note, multiple subcutaneous injections decreased B2m gene expression and B2M protein levels by ∼80% and ∼85%, respectively. These results show that mannose-conjugation with oligonucleotides is expected to help deliver oligonucleotides to macrophages and regulate gene expression in vivo, particularly in the pancreas.

Intra-abdominal ectopic bronchogenic cyst with a mucinous neoplasm harboring a GNAS mutation: A case report.

BACKGROUND: Bronchogenic cysts are congenital cysts caused by abnormal sprouting from the ventral foregut during fetal life. They usually occur in the mediastinum or lung, but there are very rare cases of ectopic bronchogenic cysts that develop in the abdominal cavity. A unique intra-abdominal ectopic bronchogenic cyst with a mucinous neoplasm that was producing carcinoembryonic antigen (CEA), harboring a GNAS mutation, is reported. The present case may contribute to clarifying the mechanism of tumorigenesis and malignant transformation of ectopic bronchogenic cysts. CASE SUMMARY: In 2007, a man in his 50s was incidentally found to have an intra-abdominal cystic mass, 8 cm in diameter. Surgical resection was recommended, but he preferred to remain under observation. In 2020, his serum CEA level increased to 26.7 ng/mL, and abdominal computed tomography showed a 15 cm × 12 cm, multifocal, cystic mass located predominantly on the lesser curvature of the stomach. Since malignancy could not be ruled out, he finally underwent surgical resection. Histologically, the cystic wall was lined by ciliated columnar epithelium, accompanied by bronchial gland-like tissue, bronchial cartilage, and smooth muscle. Part of the cyst consisted of atypical columnar epithelium with an MIB-1 index of 5% and positive for CEA. Moreover, a GNAS mutation (p.R201C) was detected in the atypical epithelium, leading to a diagnosis of an ectopic bronchogenic cyst with a low-grade mucinous neoplasm. The patient is currently undergoing outpatient follow-up without recurrence. CONCLUSION: An extremely rare case of an abdominal bronchogenic cyst with a low-grade mucinous neoplasm harboring a GNAS mutation was reported.

In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation.

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

A successful trimodality therapy for difficult-to-diagnose primary mediastinal dedifferentiated liposarcoma, which originated from the perihilar fat and invaded the right lungs.

A 40-year-old man with high fever, hemoptysis, and fatigue showed a 10-cm mass in the middle and lower lobes of the right lung on computed tomography. Histological examination of transbronchial biopsy specimens showed sheets of small round tumor cells and mild staining for CD99. Primary Ewing sarcoma was suspected, and a trimodality therapy consisting of chemotherapy, intensity-modulated radiation therapy, and right pneumonectomy with surrounding tissue resection was performed. In surgical specimens, negative outcome of NKX2.2 in immunostaining and EWSR1 rearrangement in fluorescence in situ hybridization did not support the diagnosis of Ewing sarcoma. Positive immunostaining for MDM2 and CDK4 led to a diagnosis of dedifferentiated liposarcoma, which probably originated from an adipose tissue of the right perihilar mediastinum, and then invaded the lungs. The postoperative course was uneventful, without recurrence for more than 16 months.

Successful hepatic resection for invasive Klebsiella pneumoniae large multiloculated liver abscesses with percutaneous drainage failure: A case report.

Background: Invasive Klebsiella-associated liver abscesses can progress rapidly and cause severe metastatic infections such as meningitis and hydrocephalus, which are associated with high morbidity and mortality. In patients with large multiloculated liver abscesses after failure of percutaneous drainage, rapid diagnosis of the abscess followed by hepatic resection is necessary for early recovery and to prevent severe secondary metastatic complications. Case presentation: An 84-year-old woman with a large liver abscess and in septic shock was transferred to our hospital. Abdominal CT showed multiloculated liver abscesses 15 cm in diameter in the right lobe of the liver. We first performed percutaneous liver abscess drainage. The patient was managed in the intensive care unit, as well as treated with intravenous administration of meropenem followed by cefozopran according to the antibiogram. Klebsiella pneumoniae with invasive infection was confirmed by a string test in an isolated colony of K. pneumoniae; the K1 serotype with the rmpA and magA genes was determined by polymerase chain reaction and Sanger sequencing. Additional percutaneous liver abscess drainage was performed due to initial inadequate drainage. Although the abscess had shrunk to a diameter of 8 cm after drainage in 4 weeks, the patient recovered from sepsis, but still had low-grade fever (occasionally 38°C) and continued to have symptoms of chronic inflammation with persistent hyper mucus discharge from the liver abscess. Surgical resection was chosen to prevent prolonged hospitalization and ensure early recovery. A right posterior sectionectomy of the liver, including liver abscess, was performed. The post-operative course was uneventful, with no complications, and she was discharged after 18 days. There were no signs of abscess recurrence 1 year after surgery. Conclusion: We present a case of successful hepatic resection after percutaneous drainage failure in a patient with invasive K. pneumoniae multiloculated liver abscess.

Minimal change disease soon after Pfizer-BioNTech COVID-19 vaccination.

We report on the onset of minimal change disease (MCD) presenting with anasarca after a second dose of the messenger RNA (mRNA)-based Pfizer-BioNTech vaccine against coronavirus disease 2019 (COVID-19). A 75-year-old previously healthy male was admitted with rapidly progressive anasarca and proteinuria of 7.7 g/day following the second dose. A kidney biopsy revealed MCD with nephrotic syndrome. He was treated with intravenous methylprednisolone followed by prednisolone, leading to complete remission after 35 days in the hospital. Since definite causality between the vaccine and MCD remains unclear, awareness of this potential adverse effect of mRNA vaccines is important to determine its true incidence and frequency.