Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

High serum proteinase-3 levels predict poor progression-free survival and lower efficacy of bevacizumab in metastatic colorectal cancer.

BACKGROUND: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers of their prognosis and chemotherapeutic responsiveness is necessary. This study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab therapy, in mCRC. METHODS: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer in the TCGA database. Preoperative serum PRTN3 levels were measured using an enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay. RESULTS: Serum PRTN3 levels were an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio, 2.082; 95% confidence interval, 1.118-3.647; P=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data sets showed poorer overall survival in patients with PRTN3 expression than that in patients without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first-line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poor PFS (hazard ratio, 3.027; 95% confidence interval, 1.175-7.793; P=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. The tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab therapy. CONCLUSIONS: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy, especially for bevacizumab therapy, in patients with mCRC.

The Absorb GT1 Bioresorbable Vascular Scaffold System　- 5-Year Post-Market Surveillance Study in Japan.

BACKGROUND: The 1-year clinical outcomes of the Absorb GT1 Japan post-market surveillance (PMS) suggested that an appropriate intracoronary imaging-guided bioresorbable vascular scaffold (BVS) implantation technique may reduce the risk of target lesion failure (TLF) and scaffold thrombosis (ST) associated with the Absorb GT1 BVS. The long-term outcomes through 5 years are now available.Methods and Results: This study enrolled 135 consecutive patients (n=139 lesions) with ischemic heart disease in whom percutaneous coronary intervention (PCI) with the Absorb GT1 BVS was attempted. Adequate lesion preparation, imaging-guided appropriate sizing, and high-pressure post-dilatation using a non-compliant balloon were strongly encouraged. All patients had at least 1 Absorb GT1 successfully implanted at the index procedure. Intracoronary imaging was performed in all patients (optical coherence tomography: 127/139 [91.4%] lesions) and adherence to the implantation technique recommendations was excellent: predilatation, 100% (139/139) lesions; post-dilatation, 98.6% (137/139) lesions; mean (±SD) post-dilatation pressure, 18.8±3.5 atm. At 5 years, the follow-up rate was 87.4% (118/135). No definite/probable ST was reported through 5 years. The cumulative TLF rate was 5.1% (6/118), including 2 cardiac deaths, 1 target vessel-attributable myocardial infarction, and 3 ischemia-driven target lesion revascularizations. CONCLUSIONS: Appropriate intracoronary imaging-guided BVS implantation, including the proactive use of pre- and post-balloon dilatation during implantation may be beneficial, reducing the risk of TLF and ST through 5 years.

Effects of applying high-pass filtering in pelvimetry with Martius images.

AIM: The aim of this study is to investigate whether the results of pelvimetry using original radiographic Martius images and the same images with high-pass filtering applied ("edge images") would be consistent. METHODS: A total of 30 primagravidas were included in this study. Three obstetricians independently measured the anteroposterior and transverse diameters of the pelvic inlet in the original and the edge images, recording the x- and y-coordinates of the four endpoints. A Wilcoxon signed rank sum test was performed on the coordinate data to evaluate differences between the original and edge images. RESULTS: In the analysis of all coordinate data, statistically significant differences were found in both x- and y-coordinates of the sacral promontory point (SPP). In the y-coordinate of the SPP, a statistically significant difference was found in 9 of 30 pairs of images, and in all 9 the anteroposterior diameter was shorter in the edge images compared to the original images due to the more caudal placement of the SPP. CONCLUSIONS: The coordinates of the SPP on original radiographs and their edge images were not consistent in pelvimetry using Martius images. Our results suggest that improved image contrast will allow obstetricians to better assess pelvic narrowing and cephalopelvic disproportion and even reduce radiographic dose, thereby reducing risks for pregnant women and their fetuses.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

IL-6 Levels Correlate with Prognosis and Immunosuppressive Stromal Cells in Patients with Colorectal Cancer.

BACKGROUND: The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels. METHODS: Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases. RESULTS: Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3+ and CD4+ T cells as well as FOXP3+ cells. Mass cytometry analysis showed that IL-6+ cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3highCD45RA- effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10+ cells in MDSCs and that of IL-10+ or CTLA-4+ cells in eTregs correlated with IL-6 levels. CONCLUSION: Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME.

Haemophilia and cardiovascular disease in Japan: Low incidence rates from ADVANCE Japan baseline data.

INTRODUCTION: With the increasing life expectancy of people with haemophilia, the risk of cardiovascular disease (CVD) and thrombotic events has become a growing concern. Longitudinal studies on the incidence and risk factors of CVD in this population are limited, and optimal prevention and treatment strategies are yet to be established. AIM: This study aimed to present the baseline data of a prospective longitudinal study focusing on a subset of Japanese patients with haemophilia, specifically investigated the incidence, risk factors and treatment modalities for CVD and thrombotic diseases in people aged 40 years in Japan over 10 years through the ADVANCE Japan study. METHODS: The ADVANCE Japan study is a prospective multicentre cohort study involving 600 adult individuals with haemophilia A/B aged 40 years in Japan. The primary endpoint was the incidence of CVD, with secondary endpoints encompassing anticoagulant use, mortality rates, and comparison with the general population. RESULTS: Baseline data from the 600 participants revealed that thrombotic events occurred in 13 individuals (2.2%), mostly in those with haemophilia A. Atrial fibrillation was observed in 11 participants (1.8%). Hypertension and dyslipidaemia were identified as the prevalent risk factors. Various prophylactic treatments were employed, and no severe bleeding events were observed during the study period. CONCLUSION: This study provides vital baseline data for a 10-year prospective investigation of CVD and thrombotic disease risk in people with haemophilia. These findings will contribute to refining prevention and treatment approaches and improving patients' quality of life.

Prediction of Slow-Flow Phenomenon After Stent Implantation Using Near-Infrared Spectroscopy in Patients With Acute and Chronic Coronary Syndrome.

BACKGROUND: The slow-flow phenomenon is associated with worse clinical outcomes after percutaneous coronary intervention (PCI), so our goal for this study was to see how predictive how near-infrared spectroscopy (NIRS) could be.Methods and Results: We enrolled 179 lesions from 152 patients who had de novo coronary stent implantation guided by NIRS-intravascular ultrasound (IVUS) (male: 69.1%, mean age: 74.3±11.5 years, acute coronary syndrome: 65.1%, diabetes: 42.1%). NIRS automatically determined the maximum 4-mm lipid core burden index (maxLCBI4 mm) value at pre- and post-PCI procedures. The slow-flow phenomenon was defined as the deterioration of TIMI (Thrombolysis in Myocardial Infarction) flows on angiography during the PCI procedure in the absence of mechanical obstruction. The slow-flow phenomenon occurred in 13 (7.3%) lesions, and the slow-flow phenomenon group had a significantly higher maxLCBI4 mm(740±147 vs. 471±223, P<0.001). The best maxLCBI4 mmcutoff point in both acute and chronic coronary syndrome was 578 and 480, with sensitivity of 100%, for predicting the slow-flow phenomenon. In the receiver-operating characteristics analysis, the area under the curve for acute and chronic coronary syndrome was 0.849 and 0.851, respectively. CONCLUSIONS: The results of this study support the utility of NIRS-IVUS-guided PCI for the prediction of the slow-flow phenomenon.

The usefulness of tranexamic acid for bleeding symptoms of chronic consumptive coagulopathy complicated by aortic disease: a single-institute, retrospective study of 14 patients.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail. METHODS: We evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival. RESULTS: Median age was 78.5 years (range, 66-89 years) and median observation period was 448 days (range, 0-2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4-11) and median platelet count was 64 × 109/L (range, 25-97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days. CONCLUSIONS: TXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.

Phase I study of a novel therapeutic vaccine as perioperative treatment for patients with surgically resectable hepatocellular carcinoma: The YCP02 trial.

AIM: Developing effective adjuvant therapies is essential for improving the surgical outcomes in patients with hepatocellular carcinoma (HCC). Immunotherapy against HCC has become a promising strategy; however, only approximately 30% of all HCC patients respond to immunotherapy. Previously, we generated the novel therapeutic vaccine comprising multi-human leukocyte antigen-binding heat shock protein 70/glypican-3 peptides with a novel adjuvant combination of hLAG-3Ig and poly-ICLC. We also confirmed the safety of this vaccination therapy, as well as its capacity for the effective induction of immune responses in a previous clinical trial. METHODS: In this phase I study, we administered this vaccine intradermally six times before surgery, and 10 times after surgery to patients with untreated, surgically resectable HCC (stage II to IVa). The primary end-points of this study were the safety and feasibility of this treatment. We also analyzed the resected tumor specimens pathologically using hematoxylin-eosin staining and immunohistochemistry for heat shock protein 70, glypican 3, CD8 and programmed death-1. RESULTS: A total of 20 human leukocyte antigen-matched patients received this vaccination therapy with an acceptable side-effect profile. All patients underwent planned surgery without vaccination-related delay. Immunohistochemical analyses revealed that potent infiltration of CD8+ T cells into tumors with target antigen expression was observed in 12 of 20 (60%) patients. CONCLUSIONS: This novel therapeutic vaccine was safe as perioperative immunotherapy for patients with HCC, and has the potential to strongly induce CD8+ T cells infiltration into tumors.

Current status of vaccine immunotherapy for gastrointestinal cancers.

Recent advances in tumor immunology and molecular drug development have ushered in a new era of cancer immunotherapy. Immunotherapy has shown promising results for several types of tumors, such as advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancers, and refractory Hodgkin's lymphoma. Similarly, efforts have been made to develop immunotherapies such as adoptive T-cell transplantation, peptide vaccines, and dendritic cell vaccines, specifically for gastrointestinal tumors. However, before the advent of immune checkpoint inhibitors, immunotherapy did not work as well as expected. In this article, we review immunotherapy, focusing on cancer vaccines for gastrointestinal tumors, which generally target eliciting tumor-specific CD8 + cytotoxic T lymphocytes (CTLs). We also review various vaccine therapies and describe the relationship between vaccines and adjuvants. Finally, we discuss prospects for the combination of immunotherapy with immune checkpoint inhibitors.

A novel prediction model of pancreatic fistula after pancreaticoduodenectomy using only preoperative markers.

BACKGROUND: Since clinically relevant postoperative pancreatic fistula (CR-POPF) can cause intra-abdominal hemorrhage and abscesses, leading to surgery-related deaths after pancreaticoduodenectomy (PD), its preoperative prediction is important to develop strategies for surgical procedures and perioperative management. This study aimed to establish a novel prediction model for CR-POPF using preoperative markers. METHODS: On a training set of 180 patients who underwent PD at the Yamaguchi University Hospital, a combination of CR-POPF predictors were explored using the leave-one-out method with a unique discrete Bayes classifier. This predictive model was confirmed using a validation set of 366 patients who underwent PD at the Osaka University Hospital. RESULTS: In the training set, CR-POPF occurred in 60 (33%)　of 180 patients and 130 (36%)　of 366 patients in the validation set using selected markers. In patients with pancreatic ductal adenocarcinoma (PDAC), the main pancreatic duct (MPD) index showed the highest prognostic performance and could differentiate CR-POPF with 87% sensitivity and 81% specificity among 84 patients in the training set. In the validation set, the sensitivity and specificity of the MPD index-based model for 130 PDAC samples were 93% and 87%, respectively. In patients with non-PDAC, the MPD index/body mass index (BMI) combination showed the highest prognostic performance and could differentiate CR-POPF with 84% sensitivity and 57% specificity among 96 patients in the training set. In the validation set, the sensitivity and specificity of the MPD index/BMI-based model for 236 non-PDAC samples were 85% and 53%, respectively. CONCLUSION: We developed a novel prediction model for pancreatic fistulas after PD using only preoperative markers. The MPD index and MPD index/BMI combination will be useful for CR-POPF assessment in PDAC and non-PDAC samples, respectively.

Risk Factors for Vulnerable Plaque Detected Using Near-Infrared Spectroscopy in Patients Receiving Statin Therapy with No History of Coronary Artery Disease.

Residual risk of atherosclerosis remains high despite the use of lipid-lowering therapy with statins. Near-infrared spectroscopy intravascular ultrasound imaging (NIRS-IVUS) can identify vulnerable plaque via the detection of lipid-rich plaque. This study aimed to reveal the clinical characteristics of patients with vulnerable plaque despite statin therapy.NIRS-IVUS was used to determine the maximum 4 mm Lipid Core Burden Index (MaxLCBI4 mm) values of 38 de novo culprit lesions from 32 patients with acute coronary syndrome (53%) (mean age: 73.1 ± 13.1 years) who underwent percutaneous coronary intervention after a minimum 6 months of statin therapy for primary prevention. A patient with vulnerable plaque was defined as an individual presenting at least 1 target lesion with a vulnerable plaque (MaxLCBI4 mm > 400). Overall, the average low-density lipoprotein cholesterol (LDL-C) level was 95.5 ± 27.2 mg/dL. Patients in the vulnerable plaque group were younger and had higher LDL-C, triglycerides, and non-high-density lipoprotein cholesterol (HDL-C) levels than those in the non-vulnerable plaque group. The MaxLCBI4 mm was positively correlated with LDL-C (P = 0.0002), triglycerides (P = 0.0003), and non-HDL-C (P = 0.0001). In multivariate analysis, all 3 treatable lipid components failed to show an independent relationship with the patients with vulnerable plaque. Using receiver-operating characteristics curve analysis, the cutoff points for LDL-C, triglycerides, and non-HDL-C were determined to be 78 mg/dL, 108 mg/dL, and 111 mg/dL, respectively, at MaxLCBI4 mm > 400. In conclusion, this study supports a more comprehensive and aggressive lipid-lowering therapy for the primary prevention of coronary artery disease.

[A Severe Case of Bleeding from Duodenal Invasion Due to Co-Morbid IPMC with Arcuate Ligament Syndrome and IPDA Aneurysm].

An 81-year-old man with a history of left hemiplegia due to a cerebral hemorrhage was admitted to a clinic because of tarry stools. Endoscopic findings revealed an ulcerative lesion with hemorrhage in the descending duodenum. The patient was transferred to our hospital for treatment. Because endoscopic hemostasis was impossible, interventional radiology(IVR) hemostasis was performed using coil embolization for the feeding artery. Simultaneously, angiography showed stenosis of the root of the celiac axis due to arch ligament syndrome and an aneurysm of the inferior pancreaticoduodenal artery (IPDA). Due to the risk of rebleeding, subtotal stomach-preserving pancreatoduodenectomy was performed after the patient's overall condition had stabilized. Despite dissecting the arcuate ligament, the hepatic artery flow did not improve. Hence, a direct arterial anastomosis between the middle colic artery and the gastroduodenal artery was performed. Furthermore, due to the proximity of the IPDA aneurysm to the superior mesenteric artery, IVR embolization for the IPDA aneurysm was performed on postoperative day 8, and he was transferred to a rehabilitation hospital on postoperative day 57. The pathological result was invasive intraductal papillary mucinous carcinoma(IPMC). The patient has been an outpatient with no recurrence 12 months postoperatively.

[Invasive Ductal Carcinoma within a Borderline Malignancy Phyllodes Tumor-A Case Report].

Phyllodes tumors are uncommon breast neoplasms that constitute 1-2% of breast malignancies. Invasive ductal carcinoma in the epithelial component of phyllodes tumor is very rare. When carcinoma is detected within the specimen, the management of treatment changes completely. We report a rare case of invasive ductal carcinoma arising in a giant borderline malignancy phyllodes tumor in a 51-year-old female patient. A painful 20 cm mass was found in her right breast, and a needle biopsy revealed fibroadenoma or benign phyllodes tumor, and a total mastectomy was performed. Pathological results showed that a borderline malignant phyllodes tumor coexisted with invasive ductal carcinoma. We explained that axillary surgery was necessary because invasive cancer was diagnosed after surgery, but the patient requested follow-up using images. Endocrine therapy was performed as postoperative adjuvant therapy, and the follow-up is underway without recurrence.

[A Case of Malignant Lymphoma of the Bile Duct Mimicking Cholangiocarcinoma].

During the postoperative follow-up for adrenal tumor for a 78-year-old male patient, a contrast-enhanced computed tomography scan revealed wall thickness with contrast effect in the cystic duct, enlarged lymph nodes along the ileocecal artery, and nodal shadow in the lower lobe of the left lung. First, the collected bile juice at ERC was submitted to cytology multiple times however, no malignant findings were noted. Next, a staging laparoscopy was performed; but the pathological findings of the enlarged lymph nodes and the abdominal lavage cytology showed no malignancy. A nodule in the lower lobe of the left lung was resected for diagnostic and therapeutic purposes, and the pathological diagnosis was primary adenocarcinoma of the lung. Finally the patient underwent exploratory laparotomy for diagnostic purposes. An intraoperative ultrasound- guided needle biopsy for mass lesion located in the medial section of the left liver was performed, and malignant lymphoma was suspected by the intraoperative pathological diagnosis. Cholecystectomy was performed to confirm the histological type, leading to the diagnosis of diffuse large B cell lymphoma. After surgery, the patient underwent 6 courses of rituximab plus CHOP therapy, and the bile duct stricture was improved.

[A Recurrent Colon Cancer Successfully Treated by Nivolumab plus Ipilimumab with On and Off for Preventing Exacerbation of Skin Disorders].

Here, we report a case in which nivolumab plus ipilimumab combination therapy was significantly effective for MSI-high recurrent colon cancer with acute exacerbation after 5-FU/L-OHP/CPT-11 treatment. At the end of 4 cycles of combination therapy, clinical CR was obtained on diagnostic imaging. At the end of the 2 cycles of transition from combination therapy to monotherapy, eosinophilia was observed in a quadratic function, and exacerbation of skin disorders was observed. Eosinophil counts normalized promptly after discontinuation of treatment, and skin disorders gradually improved. Two months after the discontinuation of treatment, monotherapy was restarted. After the resumption of treatment, an increase in eosinophils and worsening of skin symptoms were observed again, and stopped treatment. We report an interesting case in which immune checkpoint inhibiter were turned on and off according to eosinophil counts for preventing exacerbation of skin disorders, and for maintaining cancer remission by continuing immune checkpoint inhibitor treatment.

Immune Evasion of Hepatoma Cancer Stem-Like Cells from Natural Killer Cells.

BACKGROUND: Poor prognosis in liver cancer is due to its high frequency of intrahepatic metastasis. Cancer stem-like cells (CSLCs), which possess the properties of stemness, tumor initiation capability, and resistance to therapy, also exhibit metastatic potential. Immune surveillance plays an important role in the accomplishment of metastasis. Herein, the property of immune evasion in CSLCs was investigated. METHODS: Sphere cells were induced as CSLCs using a sphere induction medium containing neural survival factor-1. The expression of genes involved in immune evasion was determined using RNA-sequencing for sphere and parental cells followed by validation using flow cytometric analysis and ELISA. Susceptibility to natural killer (NK) cell-mediated cytotoxicity was examined by a chromium release assay. A xenograft model using BALB/c nu/nu mice was used to assess tumor growth. Gene set enrichment analysis was performed for interpreting RNA sequencing. RESULTS: The cell surface expressions of PD-L1, PD-L2, and CEACAM1 were upregulated and those of ULBP1 and MICA/MICB were downregulated in SK-sphere, CSLCs derived from SK-HEP-1, compared with that in parental cells. Levels of soluble MICA were elevated in conditioned medium from SK-sphere. Expression of HLA class I was not downregulated in SK-sphere. The susceptibilities to NK cell-mediated killing and secreted perforin were significantly lower in both CSLCs derived from SK-HEP-1 and HLE than in parental cells. Tumors formed upon inoculation of SK-sphere in immunodeficient mice harboring NK cells were larger than those formed upon inoculation of parental cells. CONCLUSION: Human hepatoma cell line-derived CSLCs may possess immune evasion properties, especially from NK cell-mediated immunity.

Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells.

BACKGROUND: Cancer stem cells (CSCs) are thought to play important roles in carcinogenesis, recurrence, metastasis, and therapy-resistance. We have successfully induced cancer stem-like sphere cells (CSLCs) which possess enhanced chemoresistance and metastatic potential. To enable the development of targeted therapy against CSLCs, we identified a gene responsible for this phenotype in CSLC. METHODS: Human hepatoma cell line SK-HEP-1 was used for CSLC induction with a unique sphere inducing medium, and HuH-7 cells were used as non-sphere forming cells in the same condition. RNA-sequencing was performed followed by validation with quantitative RT-PCR and western blotting. Knockdown experiments were done by using CRISPR-Cas9 genome-editing, and the rescue experiments were performed using the expressing plasmid vector. Chemoresistance and liver metastasis of the cells, was studied following the splenic injection of cells to severely immune deficient mice and evaluated using the MTS assay. Quantification of exosomes in the medium was done using ELISA. RESULTS: RAB3B was identified as an up-regulated gene in both CSLCs and prognostically poor hepatocellular carcinoma (HCC) by RNA-sequencing. RAB3B-KD cells showed altered CSLC phenotypes such as sphere formation, chemoresistance, and metastatic potentials, and those were rescued by RAB3B complementation. Increased exosome secretion was observed in CSLCs, and it was not observed in the RAB3B-KD cells. In addition, the RAB3B expression correlated with the expression of ABCG2, APOE, LEPR, LXN, and TSPAN13. CONCLUSION: The up regulation of RAB3B may play an important role in the chemoresistance and metastatic potential of CSLCs.

CD4 and FOXP3 as predictive markers for the recurrence of T3/T4a stage II colorectal cancer: applying a novel discrete Bayes decision rule.

BACKGROUND: We recently reported the relapse-free survival (RFS) significance of the combination of CD4+ and forkhead box P3+ (FOXP3) T-cell densities identified by immunohistochemistry in patients with stage I, II, and III colorectal cancer (CRC) who underwent curative resections. This study was designed to determine the optimal combination of markers that predict recurrence in patients with T factors of T3/T4a stage II CRC by applying a novel Bayes decision rule. METHODS: Using 137 cancer tissue specimens from T3/T4a stage II patients, 12 clinicopathologic and immune factors were analysed as predictive candidates for recurrence. RESULTS: Our study showed that the combination of low CD4+ and low FOXP3+ T-cell densities resulted in extremely poor RFS. CONCLUSIONS: Adjuvant chemotherapy may be considered for patients with a combination of low CD4+ and low FOXP3+ T-cell densities. The discovery of this new prognostic indicator is important for the appropriate management of patients undergoing curative resection for T3/T4a stage II CRC.