Leucine-rich repeat-containing G protein-coupled receptor 5 expression in lymph node metastases of colorectal cancer: Clinicopathological insights and prognostic implications.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a significant cancer stem cell marker in colorectal cancer (CRC), lacks lymph node (LN) expression studies. In this study, we identified LGR5 expression by RNAscope, a highly sensitive RNA in situ method, and analyzed its association with clinicopathological characteristics. Tissue microarrays were generated from primary tumors (PTs) and LN metastases in paraffin-embedded blocks of 38 CRC surgical resection materials. LGR5 expression by RNAscope was evaluated by dividing the expression levels into negative and positive expression. In all but two cases of LN metastasis, LGR5-positive dots were detected in tumor cells, and there was a wide range of LGR5-positive cells. More LGR5-positive dots were identified in the gland-forming region. Twenty-three cases were classified into a high LGR5-expression group, and 15 cases were classified into a low LGR5-expression group. In the high LGR5-expression group, the histological grade was lower than in the low LGR5-expression group (p = 0.0159), while necrosis was significantly more prevalent (p = 0.0326), and the tumor, node, metastasis stage was significantly lower (p = 0.0302). There was no association between LGR5 expression levels in LN metastases and LGR5 expression levels in PT tissue. LGR5 expression in LN metastases may influence prognosis. Further analysis may lead to new therapeutic strategies.

Increased mitochondria are responsible for the acquisition of gemcitabine resistance in pancreatic cancer cell lines.

Pancreatic ductal adenocarcinoma has a particularly poor prognosis as it is often detected at an advanced stage and acquires resistance to chemotherapy early during its course. Stress adaptations by mitochondria, such as metabolic plasticity and regulation of apoptosis, promote cancer cell survival; however, the relationship between mitochondrial dynamics and chemoresistance in pancreatic ductal adenocarcinoma remains unclear. We here established human pancreatic cancer cell lines resistant to gemcitabine from MIA PaCa-2 and Panc1 cells. We compared the cells before and after the acquisition of gemcitabine resistance to investigate the mitochondrial dynamics and protein expression that contribute to this resistance. The mitochondrial number increased in gemcitabine-resistant cells after resistance acquisition, accompanied by a decrease in mitochondrial fission 1 protein, which induces peripheral mitosis, leading to mitophagy. An increase in the number of mitochondria promoted oxidative phosphorylation and increased anti-apoptotic protein expression. Additionally, enhanced oxidative phosphorylation decreased the AMP/ATP ratio and suppressed AMPK activity, resulting in the activation of the HSF1-heat shock protein pathway, which is required for environmental stress tolerance. Synergistic effects observed with BCL2 family or HSF1 inhibition in combination with gemcitabine suggested that the upregulated expression of apoptosis-related proteins caused by the mitochondrial increase may contribute to gemcitabine resistance. The combination of gemcitabine with BCL2 or HSF1 inhibitors may represent a new therapeutic strategy for the treatment of acquired gemcitabine resistance in pancreatic ductal adenocarcinoma.

ARL4C is associated with epithelial-to-mesenchymal transition in colorectal cancer.

BACKGROUND: ADP-ribosylation factor-like protein 4 C (ARL4C) is a member of the ARF small GTP-binding protein subfamily. The ARL4C gene is highly expressed in colorectal cancer (CRC). ARL4C protein promotes cell motility, invasion, and proliferation. METHODS: We investigated the characteristics of ARL4C by comparing its expression at the invasion front and relationships with clinicopathological data using RNAscope, a highly sensitive RNA in situ method. RESULTS: In all cases, ARL4C expression was observed in cancer stromal cells and cancer cells. ARL4C expression in cancer cells was localized at the invasion front. In cancer stromal cells, ARL4C expression was significantly stronger in cases with high-grade tumor budding than in cases with low-grade tumor budding (P = 0.0002). Additionally, ARL4C expression was significantly increased in patients with high histological grade compared with those with low histological grade (P = 0.0227). Furthermore, ARL4C expression was significantly stronger in lesions with the epithelial-to-mesenchymal transition (EMT) phenotype compared with the non-EMT phenotype (P = 0.0289). In CRC cells, ARL4C expression was significantly stronger in cells that had the EMT phenotype compared with those with a non-EMT phenotype (P = 0.0366). ARL4C expression was significantly higher in cancer stromal cells than in CRC cells (P < 0.0001). CONCLUSION: Our analysis reinforces the possibility that ARL4C expression worsens the prognosis of patients with CRC. Further elucidation of the function of ARL4C is desired.

Impact of Early Enteral Nutrition on Delayed Gastric Emptying and Nutritional Status After Pancreaticoduodenectomy.

BACKGROUND: Although early enteral nutrition (EEN) is an accepted practice after pancreaticoduodenectomy (PD), the impact of EEN on postoperative complications or nutritional status remains unclear. We aimed to investigate the impact of EEN on delayed gastric emptying (DGE) and nutritional status after PD. METHODS: A total of 143 patients underwent PD between January 2012 and September 2020. We excluded patients who underwent a two-stage pancreatojejunostomy, in whom the enteral tube was accidentally pulled out, or with insufficient information in their medical records. The incidence of postoperative complications was compared between patients who received EEN (EEN group, n = 21) and those who did not (control group, n = 21) after propensity score matching. Univariate and multivariate analyses were performed to identify the risk factors affecting the incidence of these complications. Nutritional status was assessed at postoperative months 1, 3, and 6. RESULTS: The incidence of grade B/C DGE in the EEN group was significantly lower than that in the control group (4.8% vs. 28.6%, p = 0.03). There was no significant difference in overall morbidity, incidence of any other postoperative complications, or all-grade DGE. In multivariate analysis, EEN was associated with a reduction in the incidence of grade B/C DGE (p < 0.01). In the analysis of nutritional status, EEN was significantly associated with better nutritional status at postoperative month 1. CONCLUSION: EEN can lead to a lower clinically relevant DGE rate and better nutritional status in the early postoperative period in patients undergoing PD.

Polygenic control of the wavy coat of the NCT mouse: involvement of an intracisternal A particle insertional mutation of the protease, serine 53 (Prss53) gene, and a modifier gene.

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration.

Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14).

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.

Cutting Edge: TAK1 Safeguards Macrophages against Proinflammatory Cell Death.

TGF-ß-activated kinase 1 (TAK1) is known to play vital roles for innate and adaptive immunity; however, little is known about its potential role in limiting biological responses such as inflammation. In this study, we report that macrophage TAK1 participates in negatively regulating inflammation by restraining proinflammatory cell death. Macrophages from TAK1-deficient mice underwent cell death in response to LPS and poly(I:C), which took place in a manner dependent on TLR/TRIF-induced active Caspase8-mediated cleavage of gasdermin D, known as an executioner of pyroptosis. Likewise, TNF-α induced Caspase8-dependent gasdermin D processing following cell death in TAK1-deficient macrophages. Importantly, we demonstrated that this type of proinflammatory macrophage death is linked to susceptibility to septic shock in mice lacking TAK1 in macrophages in a TNF-α-independent fashion. Taken together, our data revealed that TAK1 acts as a signaling checkpoint to protect macrophages from unique proinflammatory cell death, ensuring the maintenance of innate immune homeostasis.

Drug resistance mechanisms in Japanese anaplastic lymphoma kinase-positive non-small cell lung cancer and the clinical responses based on the resistant mechanisms.

The treatment for anaplastic lymphoma kinase (ALK)-positive lung cancer has been rapidly evolving since the introduction of several ALK tyrosine kinase inhibitors (ALK-TKI) in clinical practice. However, the acquired resistance to these drugs has become an important issue. In this study, we collected a total of 112 serial biopsy samples from 32 patients with ALK-positive lung cancer during multiple ALK-TKI treatments to reveal the resistance mechanisms to ALK-TKI. Among 32 patients, 24 patients received more than two ALK-TKI. Secondary mutations were observed in 8 of 12 specimens after crizotinib failure (G1202R, G1269A, I1171T, L1196M, C1156Y and F1245V). After alectinib failure, G1202R and I1171N mutations were detected in 7 of 15 specimens. G1202R, F1174V and G1202R, and P-gp overexpression were observed in 3 of 7 samples after ceritinib treatment. L1196M + G1202R, a compound mutation, was detected in 1 specimen after lorlatinib treatment. ALK-TKI treatment duration was longer in the on-target treatment group than that in the off-target group (13.0 vs 1.2 months). In conclusion, resistance to ALK-TKI based on secondary mutation in this study was similar to that in previous reports, except for crizotinib resistance. Understanding the appropriate treatment matching resistance mechanisms contributes to the efficacy of multiple ALK-TKI treatment strategies.

Dissociated responses at initial computed tomography evaluation is a good prognostic factor in non-small cell lung cancer patients treated with anti-programmed cell death-1/ligand 1 inhibitors.

BACKGROUND: Dissociated responses (DR) are phenomena in which some tumors shrink, whereas others progress during treatment of patients with cancer. The purpose of the present study was to evaluate the frequency and prognosis of DR in non-small cell lung cancer (NSCLC) patients treated with anti-programmed cell death-1/ligand 1 (anti-PD-1/L1) inhibitors. METHODS: This retrospective study included NSCLC patients who received anti-PD-1/L1 inhibitor as second- or later-line treatment. We excluded patients without radiological evaluation. In patients who showed progressive disease (PD) according to the RECIST 1.1 at the initial CT evaluation, we evaluated all measurable lesions in each organ to identify DR independently of RECIST 1.1. We defined DR as a disease with some shrinking lesions as well as growing or emerging new lesions. Cases not classified as DR were defined as 'true PD'. Overall survival was compared between patients with DR and those with true PD using Cox proportional hazards models. RESULTS: The present study included 62 NSCLC patients aged 27-82 years (median: 65 years). DR and true PD were observed in 11 and 51 patients, respectively. The frequency of DR in NSCLC patients who showed PD to anti-PD-1/L1 was 17.7%. Median overall survival was significantly longer in patients with DR versus true PD (14.0 vs. 6.6 months, respectively; hazard ratio for death: 0.40; 95% confidence interval: 0.17-0.94). CONCLUSIONS: Patients with DR exhibited a relatively favorable prognosis.

Efficacy of anti-PD-1 therapy for recurrence after chemoradiotherapy in locally advanced NSC LC.

PURPOSE: Chemoradiotherapy (CRT) is the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Recently, anti-PD-1 antibody therapy became a key treatment for stage IV NSCLC as the combination of immune checkpoint inhibitors (ICIs) and platinum doublet chemotherapy. However, the efficacy and toxicity of anti-PD-1 therapy for recurrence after CRT in stage III NSCLC are not well examined. METHODS: Patients who received anti-PD-1 therapy for recurrence after CRT were identified in our clinical database. The safety and efficacy of anti-PD-1 therapy were retrospectively analyzed. RESULTS: From March 1, 2013 to April 30, 2018, there were 20 patients who received anti-PD-1 therapy for recurrence after CRT. The median duration from CRT to initial anti-PD-1 therapy was 9.3 months. 12 patients (60%) were alive and 7 patients (35%) were still receiving anti-PD-1 therapy at the data cutoff point (median follow-up, 13.5 months). The ORR for anti-PD-1 therapy was 45.0%. Median progression-free survival (PFS) and overall survival (OS) from initiation of anti-PD-1 therapy was 8.4 months and 26.2 months, respectively. PFS in patients who had a short interval from last CRT to initial anti-PD-1 therapy seemed to have better outcomes (duration from last CRT to initial anti-PD-1 therapy < 9.3 months vs. ≥ 9.3 months; median PFS, 17.0 months vs. 4.9 months). Grade 3 or 4 immune-related adverse events occurred in 5% of patients. Only grade 1 pneumonitis was observed. CONCLUSION: The efficacy of anti-PD-1 therapy for recurrence after CRT in stage III NSCLC might better than in stage IV NSCLC. The duration from CRT to initial anti-PD-1 therapy might be related to efficacy.

Barcode sequencing identifies resistant mechanisms to epidermal growth factor receptor inhibitors in circulating tumor DNA of lung cancer patients.

Most patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) will inevitably develop acquired resistance induced by treatment with EGFR tyrosine kinase inhibitors (EGFR-TKI). The mechanisms of resistance to EGFR-TKI are multifactorial, and the detection of these mechanisms is critical for treatment choices in patients who have progressed after EGFR-TKI therapy. We evaluated the feasibility of a molecular barcode method using next-generation sequencing to detect multifactorial resistance mechanisms in circulating tumor DNA and compared the results with those obtained using other technologies. Plasma samples were collected from 25 EGFR mutation-positive NSCLC patients after the development of EGFR-TKI resistance. Somatic mutation profiles of these samples were assessed using two methods of next-generation sequencing and droplet digital PCR (ddPCR). The positive rate for EGFR-sensitizing mutations was 18/25 (72.0%) using ddPCR, 17/25 (68.0%) using amplicon sequencing, and 19/25 (76.0%) using molecular barcode sequencing. Rate of the EGFR T790M resistance mutation among patients with EGFR-sensitizing mutations was shown to be 7/18 (38.9%) using ddPCR, 6/17 (35.3%) using amplicon sequencing, and 8/19 (42.1%) using molecular barcode sequencing. Copy number gain in the MET gene was detected in three cases using ddPCR. PIK3CA, KRAS and TP53 mutations were detected using amplicon sequencing. Molecular barcode sequencing detected PIK3CA, TP53, KRAS, and MAP2K1 mutations. Results of the three assays were comparable; however, in cell-free DNA, molecular barcode sequencing detected mutations causing multifactorial resistance more sensitively than did the other assays.

Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency.

Collagen is one of the most important components of the extracellular matrix that is involved in the strength of tissues, cell adhesion and cell proliferation. Mutations in several collagen and post-translational modification enzyme genes cause Ehlers-Danlos syndrome (EDS) characterized by joint and skin hyperextensibility as well as fragility of various organs. Carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase-1 (CHST14/D4ST1) is a critical enzyme for biosynthesis of dermatan sulfate, a side chain of various proteoglycans including biglycan that regulates collagen fibrils through their interaction. Mutations in CHST14 were found to cause a new form of EDS, named musculocontractural type EDS (mcEDS-CHST14). Large subcutaneous hematomas are one of the most serious complications accompanied by decreased quality of life and potential lethality. In this study, Chst14 gene-deleted mice were expected to be an animal model of the vascular abnormalities of mcEDS-CHST14. However, only limited numbers of adult mice were generated because of perinatal lethality in most Chst14 gene-deleted homozygote (Chst14-/-) mice. Therefore, we investigated the placentas of these fetuses. The placentas of Chst14-/- fetuses showed a reduced weight, alterations in the vascular structure, and ischemic and/or necrotic-like changes. Electron microscopy demonstrated an abnormal structure of the basement membrane of capillaries in the placental villus. These findings suggest that Chst14 is essential for placental vascular development and perinatal survival of fetuses. Furthermore, placentas of Chst14-/- fetuses could be a useful model for vascular manifestations in mcEDS-CHST14, such as the large subcutaneous hematomas.

Survival Benefits of Surgical Resection in Patients with Recurrent Biliary Tract Carcinoma.

BACKGROUND: Whether surgical resection for recurrent biliary tract carcinoma (BTC) prolongs survival and the patients who are most likely to benefit from such treatment remain unclear. METHODS: Among 251 patients with recurrences after the initial resection of BTC, a total of 21 patients (8.4%) underwent surgical resection for the recurrence, with a zero mortality rate. The clinicopathological features of these patients were compared with those of patients who did not undergo surgery. RESULTS: The median survival time (MST) after the first recurrence and the 5-year post-recurrent survival (PRS) rate were 19.8 months and 32.8%, respectively, for patients who underwent re-resection. Fourteen patients (66.7%) experienced second recurrences; however, none of these patients underwent further surgical resection. Surgical resection for recurrence was identified as an independent prognostic factor for survival after recurrence (hazard ratio of 0.33, 95% CI of 0.17-0.58, p < 0.001). Patients with less than three liver metastases had a significantly better PRS after surgical resection than after chemotherapy (p = 0.015). Among the patients with an isolated solitary liver metastasis, patients who underwent resection had a significantly longer MST after the first recurrence than patients receiving chemotherapy (22.8 vs. 10.9 months, p = 0.025), whereas the PRS was similar between the two groups among patients with two liver lesions. CONCLUSIONS: Surgical resection for recurrent BTC may prolong survival in highly selected patients. A hepatectomy might offer a survival benefit for patients with a solitary liver metastasis.

Differential roles of MMP-9 in early and late stages of dystrophic muscles in a mouse model of Duchenne muscular dystrophy.

Matrix metalloprotease (MMP)-9 is an endopeptidase associated with the pathogenesis of Duchenne muscular dystrophy (DMD). The precise function of MMP-9 in DMD has not been elucidated to date. We investigated the effect of genetic ablation of MMP-9 in the mdx mouse model (mdx/Mmp9(-/-)). At the early disease stage, the muscles of mdx/Mmp9(-/-) mice showed reduced necrosis and neutrophil invasion, accompanied by down-regulation of chemokine MIP-2. In addition, muscle regeneration was enhanced, which coincided with increased macrophage infiltration and upregulation of MCP-1, and resulted in increased muscle strength. The mdx/Mmp9(-/-) mice also displayed accelerated upregulation of osteopontin expression in skeletal muscle at the acute onset phase of dystrophy. However, at a later disease stage, the mice exhibited muscle growth impairment through altered expression of myogenic factors, and increased fibroadipose tissue. These results showed that MMP-9 might have multiple functions during disease progression. Therapy targeting MMP-9 may improve muscle pathology and function at the early disease stage, but continuous inhibition of this protein may result in the accumulation of fibroadipose tissues and reduced muscle strength at the late disease stage.

Effects of Adrenomedullin on Doxorubicin-Induced Cardiac Damage in Mice.

Doxorubicin (DOX) is one of the best known anticancer drugs, and is used in the treatment of lymphoma, lung cancer, stomach cancer, and a number of other cancers. However, DOX has some serious side effects, the worst being lethal heart failure. Occasionally, its side effects result in the cessation of the anticancer treatment, thus having a serious adverse influence on prognosis. Agents that can be administered as alternative prophylactics or to ameliorate the side effects of DOX will be useful in increasing the safety and efficacy of anticancer therapy. Adrenomedullin (AM) is a peptide hormone secreted by many organs, including the heart; it has an organ-protective effect, including antiapoptotic, anti-inflammatory, and antioxidative stress. Blood AM levels increase with heart failure; endogenic AM has been suggested in order to protect the heart. Furthermore, exogenous AM administration has shown therapeutic effects for heart failure in patients. However, it is unclear whether AM can protect the heart against drug-induced cardiac injury in vivo. The present study was performed in order to investigate the effects of AM on DOX-induced cardiac damage. Male BALB/c mice were treated with DOX and/or AM. Exogenous AM improved the survival ratio of DOX-treated mice. In addition, AM reduced serum lactate dehydrogenase (LDH) levels following DOX treatment. On pathological examination, AM was shown to inhibit DOX-induced cardiac tissue damage, mitochondrial abnormality, and cell death. These findings suggest that AM has a protective effect against DOX-induced cardiac damage.

Vascular endothelial adrenomedullin-RAMP2 system is essential for vascular integrity and organ homeostasis.

BACKGROUND: Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. METHODS AND RESULTS: We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. CONCLUSIONS: Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage.

Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Exploring LGR5 as a prognostic marker of extrahepatic cholangiocarcinoma: insights from expression analysis and clinical correlations.

BACKGROUND: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a cancer stem cell (CSC) marker of colorectal cancer and may be a CSC marker of other cancer types. Few studies have been conducted on LGR5 expression in extrahepatic cholangiocarcinoma (ECC). METHODS: We analyzed LGR5 expression using RNAscope, a highly sensitive RNA in situ hybridization technique. Fifty-three ECCs were selected from the medical archives at Shinshu University Hospital and analyzed using a tissue microarray. LGR5 expression levels were divided into expression and no expression groups. LGR5 expression and clinicopathological characteristics were analyzed. RESULTS: Among 25 cases, no LGR5-positive dots were identified. Among 28 cases, some LGR5-positive dots were observed in carcinoma cells, together with a wide range of LGR5-positive cells. LGR5 expression was conspicuous in glandular duct formations. Well- to moderately differentiated types showed significantly higher LGR5 expression than the poorly differentiated type (p = 0.0268). LGR5 expression was associated with good overall survival (p = 0.0219) and good disease-free survival (DFS) (p = 0.0228). High LGR5 expression was associated with well- to moderately-differentiated types, indicating a favorable prognosis. In terms of DFS, multivariate analysis showed that high LGR5 expression was an independent favorable prognostic factor (p = 0.0397). CONCLUSIONS: These findings suggest that LGR5 is a promising, novel prognostic marker.

Utilization of muscle area in an accurate prediction formula for renal function for patients with hepatocellular carcinoma.

OBJECTIVE: Accurate assessment of renal function prior to surgery for hepatocellular carcinoma is important for patient outcome, but current methods such as the estimated glomerular filtration rate (eGFR) are inadequate. We developed a new prediction formula that incorporates preoperative computed tomography (CT) imaging data to determine renal function. METHODS: We retrospectively analyzed 400 patients who underwent hepatectomy for hepatocellular carcinoma between January 2010 and December 2021. Predictors associated with renal function were identified by multivariate analysis. RESULTS: Age, sex, body height, body weight, body surface area, body mass index, serum creatinine, and muscle areas including third lumbar vertebra total muscle area (L3 TMA) determined by preoperative CT were identified as independent predictors likely to be associated with renal function. These were used to construct a new prediction formula using multiple regression analysis performed with a stepwise method: 232.2 + (-1.17 × age) + (-89.0 × serum creatinine) + (0.28 × L3 TMA). The median difference between conventional eGFR and CCr was 47.6 ml/min (range, 1.7-137.9 ml/min), while that between the new eGFR and CCr was 14.3 ml/min (range, 0.02-64.7 ml/min). Spearman rank correlation analysis revealed that the new eGFR was more positively correlated with CCr than conventional eGFR (ρ = 0.623, P < 0.05; ρ = 0.700, P < 0.05, respectively), and hence more accurately reflected renal function. CONCLUSION: A new prediction formula based on L3 TMA determined by CT is more accurate than conventional eGFR for evaluating renal function.

Long-term remnant liver volume dynamics after major hepatectomy for perihilar cholangiocarcinoma following portal vein embolization.

BACKGROUND: Portal vein embolization (PVE) followed by major hepatectomy is a common treatment strategy for patients with perihilar cholangiocarcinoma (PHCC); however, the long-term dynamics of the liver remnant volume (LRV) remain unclear. Here, we report the dynamics of the LRV in patients who underwent hepatectomy following PVE. METHODS: A total of 39 patients with PHCC who underwent right hemihepatectomy or left trisectionectomy with extrahepatic bile duct resection between 2004 and 2021 were enrolled in this study [PVE (n = 27) and non-PVE (n = 12]). Long-term remnant liver dynamics were analyzed in propensity score-matched pairs (n = 10/group). RESULTS: The LRV/future liver remnant volume (FLRV) at 1 week to 1 month after hepatectomy were smaller in the PVE group than in the non-PVE group (1.53 vs. 1.69, p = .044 and 1.52 vs 1.99, p = .003, respectively). In the non-PVE group, the LRV/FLRV ratio plateaued 1-3 months postoperatively, whereas progressive hypertrophy occurred in the PVE group, and the LRV/FLRV ratio became equal in both groups at 1 year after hepatectomy (1.96 vs. 1.97; p = .799). Multivariate analysis revealed that FLRV/total liver volume (TLV) ≤ 0.43 was the only independent predictor of LRV/FLRV ≥1.9 at 1 year after hepatectomy (odds ratio:5.345, 95% confidence interval:1.210-23.615; p = .027). CONCLUSION: Although the long-term LRV was nearly equal in both groups, short-term LRV hypertrophy was lower in the PVE group than in the non-PVE group.