Diagnostic guide for immune checkpoint inhibitor-induced liver injury.

With the widespread use of immune checkpoint inhibitors (ICIs), liver injury (ICI-induced liver injury) as an immune-related adverse event has become a major concern in clinical practice. Because severe cases of liver injury require administration of corticosteroids, a comprehensive evaluation is crucial, including clinical course, blood and imaging tests, and if necessary, pathological examination through liver biopsy. As with liver injury induced by other drugs, classification of injury type by R-value is useful in deciding treatment strategies for ICI-induced liver injury. Histologically, the most representative feature is an acute hepatitis-like hepatocellular injury, characterized by diffuse lobular inflammation accompanied by CD8-positive T lymphocytes. Another condition that can cause liver injury during ICI treatment is cholangitis accompanied by non-obstructive bile duct dilatation and bile duct wall thickening. Many cases of ICI-induced cholangitis are classified as non-hepatocellular injury type, and they have been reported to respond poorly to corticosteroids. It is essential that gastroenterologists/hepatologists and doctors in various departments work in cooperation to develop a system that achieves early diagnosis and appropriate treatment of ICI-induced liver injury.

Impact of Nutritional Status on Neutrophil-to-Lymphocyte Ratio as a Predictor of Efficacy and Adverse Events of Immune Check-Point Inhibitors.

The neutrophil -to-lymphocyte ratio (NLR) is useful for predicting the effectiveness of treatment with immune checkpoint inhibitors (ICIs) and immune-related adverse events (irAEs). Because a growing body of evidence has recently shown that the number of lymphocytes that comprise NLR fluctuates according to nutritional status, this study examined whether the usefulness of NLR varies in ICI treatment due to changes in nutritional status. A retrospective analysis was performed on 1234 patients who received ICI treatment for malignant tumors at our hospital. Progression-free survival (PFS) was significantly prolonged in patients with NLR < 4. Multivariate analysis revealed that the factors associated with the occurrence of irAE were NLR < 4 and the use of ipilimumab. However, when limited to cases with serum albumin levels <3.8 g/dL, lymphocyte counts significantly decreased, and the associations between NLR and PFS and between NLR and irAE occurrence disappeared. In contrast, when limited to the cases with serum albumin levels ≥3.8 g/dL, the associations remained, with significantly prolonged PFS and significantly increased irAE occurrence at NLR < 4. NLR may be a good predictive tool for PFS and irAE occurrence during ICI treatment when a good nutritional status is maintained.

Predictive factors for transition to conversion therapy in hepatocellular carcinoma using atezolizumab plus bevacizumab.

BACKGROUND: To identify predictive factors associated with successful transition to conversion therapy following combination therapy with atezolizumab and bevacizumab in the treatment of unresectable hepatocellular carcinoma (HCC). METHODS: In total, 188 patients with HCC, who received atezolizumab plus bevacizumab combination therapy as the first-line chemotherapy, were studied. Patients who achieved complete response (CR) with systemic chemotherapy alone were excluded. Clinical factors possibly linked to successful transition to conversion therapy and the achievement of cancer-free status were identified. RESULTS: Fifteen (8.0%) patients underwent conversion therapy. In the conversion group, there was a significantly higher proportion of patients with Barcelona Clinic Liver Cancer (BCLC) stage A or B (73.3% versus [vs.] 45.1%; p = .03) and tended to have lower Child-Pugh scores and alpha-fetoprotein levels. Multivariate analysis revealed that BCLC stage was a predictive factor for the implementation of conversion therapy (A or B; odds ratio 3.7 [95% CI: 1.1-13]; p = .04). Furthermore, 10 (66.7%) patients achieved cancer-free status and exhibited a smaller number of intrahepatic lesions at the start of treatment (3.5 vs. 7; p < .01), and a shorter interval between systemic chemotherapy induction and conversion therapy (131 vs. 404 days; p < .01). In addition, the rate of achieving cancer-free status by undergoing surgical resection or ablation therapy was significantly higher (p = .03). CONCLUSION: BCLC stage was the sole predictive factor for successful transition to conversion therapy when using combination therapy with atezolizumab and bevacizumab to treat HCC. Furthermore, a small number of intrahepatic lesions and early transition to conversion therapy were associated with the achievement of cancer-free status.

A Luciferase Reporter Assay to Detect Cellular Hypoxia In Vitro.

Hypoxia is a hallmark of ischemic cardiovascular diseases and solid malignant tumors. Cellular hypoxia induces numerous physiological and pathological processes, including hematopoiesis, angiogenesis, metabolic changes, cell growth, and apoptosis. Hypoxia-inducible factor-1 (HIF-1) binds to hypoxia response elements (HREs) to selectively induce the expression of various genes in response to hypoxia. Therefore, HREs have been used to develop hypoxia-targeted gene therapy.More than 70 pairs of HREs and hypoxia-inducible genes have been identified. The hypoxia-induced gene expression levels vary among HRE sequences depending on the number of HRE copies and oxygen levels. Most known HREs have not yet been thoroughly studied. Recent studies have revealed that the HRE-mediated effects of hypoxia are cell line-dependent. Herein we describe an in vitro method to investigate gene activation levels and characteristics based on varying the copy number of HREs in response to cellular hypoxia. We explain how to clone HREs into luciferase reporter constructs in the sense, antisense, and dual directions to measure luciferase expression for functional analyses.

Radionuclide Reporter Imaging to Visualize Tumor Hypoxia Ex Vivo and In Vivo.

In vitro studies using cell culture, including three-dimensional cultures without the involvement of tumor vessels, have limitations in simulating complex intratumoral hypoxic conditions in live subjects. To generate experimental hypoxic conditions closer to those observed in humans in clinical settings, in vivo studies are necessary. In addition, visible light generated via bioluminescence and fluorescence is generally unsuitable for in vivo experiments because of low tissue penetration. Furthermore, near-infrared light (NIR), which has the highest tissue penetration among lights of different wavelengths, cannot be assessed precisely in vivo because of the difficulty in correcting tissue absorption and scatter. For in vivo quantitative analyses, imaging modalities that use high tissue-penetrating signals, such as computed tomography (CT) using X-rays, radionuclide imaging using γ-rays, and magnetic resonance imaging (MRI) using electromagnetic waves, are ideal.Therefore, as an advanced protocol for this research purpose, we provide ex vivo and in vivo methods to investigate the genetic response of multiple copies of hypoxia response elements (HREs) to tumor hypoxia in terms of intensity and intratumoral distribution using a human sodium/iodide symporter (hNIS) reporter gene and radionuclide reporter probes (radioiodine and its chemical analog Tc-99m) based on our previous research. This protocol includes cloning an hNIS reporter construct with multiple copies of HREs, establishing stable cell lines of the reporter construct, preparing a mouse subcutaneous xenograft model, and evaluating the genetic response of multiple HREs to tumor hypoxia using digital autoradiography (ARG) ex vivo and using single-photon emission computed tomography (SPECT) or positron emission tomography (PET) in vivo.

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Disease Progression-Related Markers for Aged Non-Alcoholic Fatty Liver Disease Patients.

Liver fibrosis is an important phenomenon in non-alcoholic fatty liver disease (NAFLD) progression. Standard markers reflecting liver fibrosis, including the FIB-4 index, increase with age. This study aimed to identify fibrosis progression-related markers that are diagnostically beneficial even in aged individuals. Serum levels of pro- and anti-inflammatory cytokines were measured by multiple enzyme-linked immunosorbent assay. Two standard NAFLD or fibrosis progression-related markers - the FIB-4 index and APRI score - were analyzed along with cytokine levels to define the best approach to discriminate advanced fibrosis. Ninety-eight NAFLD patients were enrolled: 59 and 39 patients with fibrosis stages 1-2 and 3-4 respectively. In addition to the FIB-4 index and APRI score, the following factors showed significant differences between stages 1-2 and stages 3-4 in a multivariate analysis: platelet counts, IP-10, and RANTES. The fibrosis stage, FIB-4, APRI, PDGF-BB, and RANTES were related to the prognosis. In aged patients, IP-10, GM-CSF, and RANTES differed between stages 1-2 and stages 3-4. FIB-4 and APRI were beneficial for their correlation with fibrosis. However, to stratify either young or elderly advanced fibrosis patients, and to identify patients likely to have a bad outcome, RANTES was the best marker.

The prediction of early progressive disease in patients with hepatocellular carcinoma receiving atezolizumab plus bevacizumab.

BACKGROUND AND AIMS: The IMbrave 150 trial revealed the usefulness of atezolizumab plus bevacizumab therapy in patients with unresectable hepatocellular carcinoma (HCC), making it now considered the first-line systemic chemotherapy agent for HCC. The present study investigated factors associated with early tumor progression of atezolizumab plus bevacizumab in patients with advanced HCC in real-world clinical practice. METHODS: A total of 184 HCC patients who received atezolizumab plus bevacizumab therapy were studied. We investigated the frequency of early progressive disease (e-PD; PD within 9 weeks) and analyzed the risk factors for e-PD. RESULTS: There were 47 patients (25.5%) diagnosed as e-PD. Patients with e-PD had a worse performance status (PS) and albumin-bilirubin (ALBI) and Child-Pugh (C-P) scores and a significantly higher rate of a systemic therapy than those with non-e-PD. A multivariate analysis showed that PS ≥1 (odds ratio [OR] = 4.5, 95% confidence interval [CI] = 1.9-10, p < 0.001), ALBI score ≥-2.30 (OR = 2.1, 95% CI = 1.0-4.5, p = 0.044) and the history of a systemic therapy (OR = 3.0, 95% CI = 1.4-6.4, p = 0.0038) were significant and independent determinants of e-PD. When examining the liver function trends in e-PD patients, the ALBI scores at 3 and 6 weeks after starting therapy were significantly higher than before the treatment (p < 0.001). CONCLUSIONS: The liver function and systemic therapy are useful predictors of e-PD in HCC patients treated with atezolizumab plus bevacizumab in real-world clinical practice.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy.

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.

Oxidative stress-related markers as prognostic factors for patients with primary sclerosing cholangitis in Japan.

BACKGROUND/PURPOSE: Primary sclerosing cholangitis (PSC) is a rare chronic liver disease. The mechanisms and prediction of PSC progression are unclear. Recent investigations have shown that general conditions, such as oxidative stress, affect the course of chronic diseases. We investigated the clinical course and oxidative stress-related condition of PSC to determine prognostic factors. METHODS: We recruited 58 patients with PSC (mean age; 37.4 years, mean observation period; 1382 days) who visited our department from 2003 to 2021. Clinical characteristics were investigated to define prognostic factors. Oxidative stress status was evaluated using two types of markers: an oxidative stress marker (serum reactive oxygen metabolite; dROM) and an antioxidant marker (serum OXY adsorbent test; OXY). RESULTS: The revised Mayo risk, Child-Pugh, model for end-stage liver disease-sodium (MELD-Na) scores or fibrosis-related FIB-4 index significantly predicted poor overall survival. High intestinal immunoglobulin A (IgA) levels predicted poor survival. Among patients with high and intermediate revised Mayo risk scores, those with physiologically high dROM levels showed better survival than those with lower dROM levels. In this population, dROM was negatively correlated with AST and IgA, which are both correlated with survival. CONCLUSIONS: High and intermediate revised Mayo risk score group predicted a poor clinical course in PSC. Additionally, the Child-Pugh score, MELD-Na score, FIB-4 index, and serum IgA were significantly correlated with survival. In patients with high and intermediate revised Mayo risk scores, physiologically high oxidative stress status correlated with low IgA levels and a good prognosis.

Diagnostic accuracy of frozen section biopsy for early gastric cancer extent during endoscopic submucosal dissection: a prospective study.

BACKGROUND: Accurate diagnosis of the lateral extent of early gastric cancer during endoscopic submucosal dissection (ESD) is crucial to achieve negative resection margins. Similar to intraoperative consultation with a frozen section in surgery, rapid frozen section diagnosis with endoscopic forceps biopsy may be useful in assessing tumor margins during ESD. This study aimed to evaluate the diagnostic accuracy of frozen section biopsy. METHODS: We prospectively enrolled 32 patients undergoing ESD for early gastric cancer. Biopsy samples for the frozen sections were randomly collected from fresh resected ESD specimens before formalin fixation. Two different pathologists independently diagnosed 130 frozen sections as "neoplasia," "negative for neoplasia," or "indefinite for neoplasia," and the frozen section diagnosis was compared with the final pathological results of the ESD specimens. RESULTS: Among the 130 frozen sections, 35 were from cancerous areas, and 95 were from non-cancerous areas. The diagnostic accuracies of the frozen section biopsies by the two pathologists were 98.5 and 94.6%, respectively. Cohen's kappa coefficient of diagnoses by the two pathologists was 0.851 (95% confidence interval: 0.837-0.864). Incorrect diagnoses resulted from freezing artifacts, a small volume of tissue, inflammation, the presence of well-differentiated adenocarcinoma with mild nuclear atypia, and/or tissue damage during ESD. CONCLUSIONS: Pathological diagnosis of frozen section biopsy is reliable and can be applied as a rapid frozen section diagnosis for evaluating the lateral margins of early gastric cancer during ESD.

Cryopreservation of tissues by slow-freezing using an emerging zwitterionic cryoprotectant.

Cryopreservation of tissues is a tough challenge. Cryopreservation is categorized into slow-freezing and vitrification, and vitrification has recently been recognized as a suitable method for tissue cryopreservation. On the contrary, some researchers have reported that slow-freezing also has potential for tissue cryopreservation. Although conventional cryoprotectants have been studied well, some novel ones may efficiently cryopreserve tissues via slow-freezing. In this study, we used aqueous solutions of an emerging cryoprotectant, an artificial zwitterion supplemented with a conventional cryoprotectant, dimethyl sulfoxide (DMSO), for cell spheroids. The zwitterion/DMSO aqueous solutions produced a better cryoprotective effect on cell spheroids, which are the smallest units of tissues, compared to that of a commercial cryoprotectant. Cryopreservation with the zwitterion/DMSO solutions not only exhibited better cell recovery but also maintained the functions of the spheroids effectively. The optimized composition of the solution was 10 wt% zwitterion, 15 wt% DMSO, and 75 wt% water. The zwitterion/DMSO solution gave a higher number of living cells for the cryopreservation of mouse tumor tissues than a commercial cryoprotectant. The zwitterion/DMSO solution was also able to cryopreserve human tumor tissue, a patient-derived xenograft.

Inflammatory cytokine-enriched microenvironment plays key roles in the development of breast cancers.

As the incidence of breast cancer continues to increase, it is critical to develop prevention strategies for this disease. Inflammation underlies the onset of the disease, and NF-κB is a master transcription factor for inflammation. Nuclear factor-κB (NF-κB) is activated in a variety of cell types, including normal epithelial cells, cancer cells, cancer-associated fibroblasts (CAFs), and immune cells. Ductal carcinoma in situ (DCIS) is the earliest stage of breast cancer, and not all DCIS lesions develop into invasive breast cancers (IBC). Currently, most patients with DCIS undergo surgery with postoperative therapy, although there is a risk of overtreatment. In BRCA mutants, receptor activator of NF-κB (RANK)-positive progenitors serve as the cell of origin, and treatment using the RANK monoclonal antibody reduces the risk of IBC. There is still an unmet need to diagnose malignant DCIS, which has the potential to progress to IBC, and to establish appropriate prevention strategies. We recently demonstrated novel molecular mechanisms for NF-κB activation in premalignant mammary tissues, which include DCIS, and the resultant cytokine-enriched microenvironment is essential for breast cancer development. On the early endosomes in a few epithelial cells, the adaptor protein FRS2ß, forming a complex with ErbB2, carries the IκB kinase (IKK) complex and leads to the activation of NF-κB, thereby inducing a variety of cytokines. Therefore, the FRS2ß-NFκB axis in the inflammatory premalignant environment could be targetable to prevent IBC. Further analysis of the molecular mechanisms of inflammation in the premalignant microenvironment is necessary to prevent the risk of IBC.

History of Transcatheter Arterial Chemoembolization Predicts the Efficacy of Hepatic Arterial Infusion Chemotherapy in Hepatocellular Carcinoma Patients.

This study sought to identify factors that are predictive of a therapeutic response to hepatic arterial infusion chemotherapy (HAIC) by focusing on the number of prior transcatheter arterial chemoembolization (TACE) sessions. To determine the parameters predicting a good response to HAIC, we retrospectively analyzed 170 patients with hepatocellular carcinoma (HCC) who received HAIC regimens comprising low-dose cisplatin combined with 5-fluorouracil (LFP) or cisplatin (CDDP) for the first time. In both the LFP and CDDP regimens, the response rates were significantly lower in patients with three or more prior TACE sessions than in those with two or fewer prior TACE sessions (LFP 57% versus 28%; p=0.01, CDDP 27% versus 6%; p=0.01). Multivariable logistic regression analysis revealed that the number of prior TACE sessions (≥ 3) was significantly associated with non-responder status (odds ratio 4.17, 95% Confidence Interval (CI) 1.76-9.86) in addition to the HAIC regimen. Multivariable analysis using the Cox proportional hazards model revealed that a larger number of prior TACE sessions (≥ 3) was a significant risk factor for survival (hazard ratio 1.60, 95% CI 1.12-2.29) in addition to Child-Pugh class, serum alpha-fetoprotein concentration, and maximum diameter of HCC. HCC patients who receive fewer prior TACE sessions (≤ 2) were found to be better responders to HAIC.

Study Protocol for a Trial: A Single-Arm, Open-Labeled Study Evaluating Transcatheter Arterial Embolization Plus Everolimus Combination Therapy for Patients With Liver Metastasis of Gastroenteropancreatic Neuroendocrine Tumors.

Background: The number of patients with non-functional neuroendocrine tumors (NETs) has increased recently, and the rate of liver metastasis of NETs is about 20% in patients at the first diagnosis. Transcatheter arterial embolization (TAE) and everolimus are therapies with reported efficacy, but few reports have described their combined treatment. We therefore aim to evaluate the efficacy and safety of combination therapy with everolimus and TAE in patients with liver metastasis of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) in a prospective study. Methods: We design a single-arm, open-label, prospective study to evaluate the efficacy and safety of combination therapy with everolimus and TAE in patients with liver metastases of GEP-NETs. The study started in June 2021 at Okayama University Hospital and is expected to enroll 18 patients over a 2-year period. Discussion: This study is a prospective study investigating a new treatment method for a rare disease called GEP-NETs. We may obtain useful information that contributes to the treatment guidelines in this study. However, NET is a rare disease, and although the number of cases is statistically established, it may not be possible to accurately assess causality.TRIAL REGISTRATION NUMBER: jRCT1061210015.

Pathogenesis of Severe Liver Injury in Patients with Anorexia Nervosa: A Report of Two Cases and a Literature Review.

Anorexia nervosa (AN) can cause severe protein energy malnutrition and the consequent development of various organ disorders. AN is known to cause hepatic complications. We report two cases of starvation and refeeding-induced liver injury in patients with AN, and review the literature on the hepatic complications of AN. Acute liver injury can be induced by both starvation and refeeding, although the underlying pathomechanisms and management of liver injury differ between these two conditions. Clinicians should carefully identify the clinical features to ensure an accurate diagnosis and appropriate management of these conditions.

Public attitudes in the clinical application of genome editing on human embryos in Japan: a cross-sectional survey across multiple stakeholders.

Recent advances in genome editing technology are accompanied by increasing public expectations on its potential clinical application, but there are still scientific, ethical, and social considerations that require resolution. In Japan, discussions pertaining to the clinical use of genome editing in human embryos are underway. However, understanding of the public's sentiment and attitude towards this technology is limited which is important to help guide the debate for prioritizing policies and regulatory necessities. Thus, we conducted a cross-sectional study and administered an online questionnaire across three stakeholder groups: the general public, patients and their families, and health care providers. We received responses from a total of 3,511 individuals, and the attitudes were summarized and compared among the stakeholders. Based on the distribution of responses, health care providers tended to be cautious and reluctant about the clinical use of genome editing, while patients and families appeared supportive and positive. The majority of the participants were against the use of genome editing for enhancement purposes. Participants expressed the view that clinical use may be acceptable when genome editing is the fundamental treatment, the risks are negligible, and the safety of the technology is demonstrated in human embryos. Our findings suggest differences in attitudes toward the clinical use of genome editing across stakeholder groups. Taking into account the diversity of the public's awareness and incorporating the opinion of the population is important. Further information dissemination and educational efforts are needed to support the formation of the public's opinion.

A case of focal nodular hyperplasia with hepatic failure treated with liver transplantation.

Focal nodular hyperplasia (FNH) is a benign nodular lesion, but because of its feature of portal tract vessel abnormality, it may induce portal hypertension. A 27-year-old woman was admitted with a fever. A large nodule with satellite lesions was found in the liver and cotton wool-like feature of arteries were detected on angiography. Technetium galactosyl serum albumin scintigraphy and diagnostic laparoscopy showed that the tumor site was functional, while the surrounding area was a non-functional fibrotic area. A biopsy specimen indicated that the nodular lesion was an FNH-like lesion. She experienced several instances of variceal rupture and suffered liver failure, receiving liver transplantation. The excised liver showed a centrally scarred area in the nodule, indicating that the diagnosis was FNH. We herein report this case as a rare case of FNH that progressed to liver failure.

The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.