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The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-gamma-STAT1 cascade augmented NK2R expression in CD8(+) T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8(+) T cells. IFN-gamma stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8(+) T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-gamma and granzyme B production by CD8(+) T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and I kappa B alpha degradation in activated CD8(+) T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-gamma-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-gamma-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8(+) T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.

Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) developed risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation.HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisulfite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We investigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model.Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in ≥2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that ≥2 methylated genes was an independent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS.Our proposed stratification system considers individual risk in HB and may improve patient clinical management.

Gemcitabine/cisplatin (GC) combination therapy has been the standard palliative chemotherapy for patients with advanced biliary tract cancer (BTC). No randomized clinical trials have been able to demonstrate the survival benefit over GC during the past decade. In our previous phase II trial, adding S-1 to GC (GCS) showed promising efficacy and we aimed to determine whether GCS could improve overall survival compared with GC for patients with advanced BTC.We performed a mulitcenter, randomized phase III trial across 39 centers. Enrolled patients were randomly allocated (1:1) to either the GCS or GC arm. The GCS regimen comprised gemcitabine (1000 mg/mBetween July 2014 and February 2016, 246 patients were enrolled. The median OS and 1-year OS rate were 13.5 months and 59.4% in the GCS arm and 12.6 months and 53.7% in the GC arm, respectively (hazard ratio [HR] 0.79, 90% confidence interval [CI]: 0.628-0.996; P = .046 [stratified log-rank test]). Median PFS was 7.4 months in the GCS arm and 5.5 months in the GC arm (HR 0.75, 95% CI: 0.577-0.970; P = .015). RR was 41.5% in the GCS arm and 15.0% in the GC arm. Grade 3 or worse AEs did not show significant differences between the two arms.GCS is the first regimen which demonstrated survival benefits as well as higher RR over GC in a randomized phase III trial and could be the new first-line standard chemotherapy for advanced BTC. To exploit the advantage of its high RR, GCS is now tested in the neoadjuvant setting in a randomized phase III trial for potentially resectable BTC.

Anticancer drugs for double cancers are selected based on their therapeutic effects on the target cancer, but there are insufficient data on the effects of anticancer drugs on comorbid cancer. We investigated the effect of chemotherapy on comorbid cancer in patients with simultaneous double cancers.The subjects of this retrospective study were 51 patients with simultaneous double cancers at the time of receiving systemic chemotherapy. We evaluated the types of anticancer drugs used for double cancers, the therapeutic effects on targeted and comorbid cancers, and prognoses.Disease control was achieved for 90.9% of the target cancers and 90.7% of the comorbid cancers. The prognosis was significantly better when the disease was controlled, not only in the target cancer but also in the comorbid cancer.Physicians treating double cancers should develop treatment strategies focusing not only on the treatment for advanced cancer, but also on the course of comorbidities and the therapeutic effects of anticancer drugs. This study is important because it presents new possibilities to expand the indications for anticancer drugs, while allowing unnecessary clinical research to be avoided.

The purpose of this study was to elucidate the effects of the difference of initial graft tension on the femorotibial relationship on an axial plane and its chronological change following anatomical anterior cruciate ligament (ACL) reconstruction.A total of 63 patients who underwent anatomical ACL reconstruction were included in this study. The graft was fixed at full knee extension with manual maximum (higher graft tension; group H) and 80 N (lower graft tension; group L) pulls in 31 and 32 patients, respectively. The femorotibial positional relationship in axial computed tomography at 1 week and 1 year postoperatively were retrospectively evaluated. The side-to-side differences (SSDs) and the amount of changes of SSDs over 1 year were compared between groups.The SSDs of the external rotational angle of the tibia in group H were significantly larger than those in group L at postoperative 1 week (2.7 ± 3.9° vs. 0.3 ± 3.3°; P 0.01). The amount of internal rotational changes of SSDs of the internal-external rotational angles over 1 year in group H was significantly larger than that in group L (-3.6 ± 3.9° vs. - 0.3 ± 2.7°; P 0.01). No significant differences were observed on the anterior-posterior translation distance and medial-lateral shift distance.The application of higher initial graft tension resulted in excessive external rotation of the tibia to the femur at 1 week postoperatively in anatomical ACL reconstruction, and the excessive early external tibial rotation had resolved over 1 year.

The development of inflammatory bowel diseases is thought to be multifactorial, but the exact steps in pathogenesis are poorly understood. In this study, we investigated involvement of the activation of STAT1 signal pathway in the pathogenesis of an acute colitis model.A dextran sulfate sodium-induced acute colitis model was established by using wild-type C57BL/6 mice and STAT1-deficient mice. Disease indicators such as body weight loss and clinical score, induction of cytokines, chemokines, and inflammatory cells were evaluated in the acute colitis model.Disease state was significantly improved in the acute colitis model using STAT1-deficient mice compared with wild-type mice. The induction of Ly6c-highly expressing cells in colorectal tissues was attenuated in STAT1-deficient mice. IL-6, CCL2, and CCR2 gene expressions in Ly6c-highly expressing cells accumulated in the inflamed colon tissues and were significantly higher than in Ly6c-intermediate-expressing cells, whereas TNF-α and IFN-α/β gene expression was higher in Ly6c-intermediate-expressing cells. Blockade of CCR2-mediated signaling significantly reduced the disease state in the acute colitis model.Two different types of Ly6c-expressing macrophages are induced in the inflamed tissues through the IFN-α/β-STAT1-mediated CCL2/CCR2 cascade and this is associated with the pathogenesis such as onset, exacerbation, and subsequent chronicity of acute colitis.

To ensure that highly advanced hepatobiliary-pancreatic surgery (HBPS) is performed safely, the Japanese Society of Hepato-Biliary-Pancreatic Surgery (JSHBPS) board certification system for expert surgeons established a safety committee to monitor surgical safety.We investigated postoperative mortality rates based on summary reports of numbers and outcomes of highly advanced HBPS submitted annually by the board-certified training institutions from 2012 to 2019. We also analyzed summary reports on mortality cases submitted by institutions with high 90-day post-HBPS mortality rates and recommended site visits and surveys as necessary.Highly advanced HBPS was performed in 121 518 patients during the 8-year period. Thirty-day mortality rates from 2012 to 2019 were 0.92%, 0.8%, 0.61%, 0.63%, 0.70%, 0.59%, 0.48%, and 0.52%, respectively (P .001). Ninety-day mortality rates were 2.1%, 1.82%, 1.62%, 1.28%, 1.46%, 1.22%, 1.19%, and 0.98%, respectively (P .001). Summary reports were submitted by 20 hospitals between 2015 and 2019. Mortality rates before and after the start of report submission and audit were 5.72% and 2.79%, respectively (odds ratio 0.690, 95% confidence interval 0.487-0.977; P = .037).Development of a system for designation of board-certified expert surgeons and safety management improved the mortality rate associated with highly advanced HBPS.

A tibial cut with the native posterior tibial slope (PTS) is a theoretical prerequisite in bicruciate-retaining total knee arthroplasty (BCRTKA) to regain physiological knee kinematics. The present study reveals tibial morphological risk factors of trauma to the posteromedial structures of the knee during tibial bone resection in BCRTKA.Fifty patients undergoing BCRTKA for varus knee osteoarthritis were analyzed. A three-dimensional tibial bone model was reconstructed using a computed tomography-based preoperative planning system, and the coronal tibial slope (CTS) and medial PTS (MPTS) were measured. Then, we set the simulated tibial cutting plane neutral on the coronal plane, posteriorly inclined in accordance with the MPTS on the sagittal plane, and 9 mm below the surface of the subchondral cortical bone (i.e., 11 mm below the surface of the cartilage) of the lateral tibial plateau. The association between the tibial morphology and the distance from the simulated cutting plane to the semimembranosus (SM) insertion (Dsm) was analyzed.Of the 50 patients, 19 (38%) had negative Dsm values, indicating a cut into the SM (namely, below the posterior oblique ligament) insertion. The MPTS was negatively correlated with Dsm (r = -0.396, p = 0.004), whereas the CTS was positively correlated with Dsm (r = 0.619, p 0.001). On multivariate linear regression analysis, the MPTS and CTS were independent predictors of Dsm.In the setting of tibial cuts reproducing the native MPTS in BCRTKA, patients with larger PTS and smaller CTS had more risk of trauma to the posteromedial structures.

Objectives This study aimed to investigate the trend of joint destruction patterns on knee radiographs of patients with rheumatoid arthritis (RA) undergoing total knee arthroplasty (TKA) over the past 16 years. Method Medial joint space, lateral joint space, medial spur area, lateral spur area (L-spur), and femoro-tibial angle were obtained from 831 preoperative knee radiographs of patients with RA who underwent TKA between 2006 and 2021 using software capable of automatic measurements. Non-hierarchical clustering was performed based on these five parameters. Trends in the five individual radiographic parameters and the ratio of each cluster were investigated during the target period. Moreover, clinical data from 244 cases were compared among clusters to identify factors associated with this trend. Results All parameters, except for L-spur, showed significant increasing trends from 2006 to 2021. The radiographs were clustered into groups according to the characteristic pattern of radiographic findings: cluster 1 (conventional RA type), with bicompartmental joint space narrowing (JSN), less spur formation, and valgus alignment; cluster 2 (osteoarthritis type), with medial JSN, medial osteophytes, and varus alignment; and cluster 3 (less destructive type), with mild bicompartmental JSN, less spur formation, and valgus alignment. The ratio of cluster 1 showed a significantly decreasing trend contrary to the significantly increasing trend in clusters 2 and 3. The DAS28-CRP of cluster 3 was higher than those of clusters 1 and 2. Conclusions Radiographs of TKA recipients with RA are increasingly presenting osteoarthritic features in recent decades. Key Points• Using automated measurement software, morphological parameters were measured from radiographs of 831 patients with rheumatoid arthritis who had undergone TKA in the past 16 years.• Cluster analysis based on the radiographic parameters revealed that the radiographs of patients with end-stage knee arthritis requiring total knee arthroplasty were classified into three groups.• In patients with rheumatoid arthritis who have undergone total knee arthroplasty in the past 16 years, the proportion of clusters with features of osteoarthritis and difficult-to-treat rheumatoid arthritis has increased, while the proportion of conventional rheumatoid arthritis has decreased.

Mammals are thermostatic animals and can regulate their body temperature within a precise range, irrespective of ambient temperature conditions. However, the mechanisms of how a body temperature is controlled in cold environments are still unclear. Here, we report that RNA binding motif protein 3 (RBM3) is crucial for maintaining body temperature in response to cold stimulation. RBM3, the RNA binding protein that stabilizes the target mRNA, is one of the significant proteins induced by cold stimulation. The Rbm3 knockout (Rbm3-/-) juvenile mice are sensitive to cold exposure and cannot maintain their body temperature. Notably,Rbm3-/-mice decreased the expression level of UCP1 and UCP3, pivotal for thermogenesis in brown adipose tissue (BAT) and skeletal muscle, respectively. We also found that a defect in RBM3 increases the size of lipid droplets in the BAT. Additionally, UCP1 expression is canceled by cold stimulation inRbm3-/-BAT. On the other hand, RBM3 deficiency has little effect on cold-induced UCP3 expression in skeletal muscle. These data suggest that RBM3 prevents body temperature reduction induced by cold stimulation via expressing UCP1 in BAT. Our findings provide a possibility that RBM3 is a crucial regulator of thermogenesis in juvenile mice with unstable body temperature regulation.

PNPLA8, one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. However, little is known about its role in brain development. Here, we report 12 individuals from 10 unrelated families with biallelic ultra-rare variants inPNPLA8presenting with a wide spectrum of clinical features ranging from developmental and epileptic-dyskinetic encephalopathy (DEDE) to progressive movement disorders. Complete loss of PNPLA8 was associated with the severe end of the spectrum, showing DEDE manifestations and congenital or progressive microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 reduced the number of basal radial glial cells (bRGCs) and upper-layer neurons. By spatial transcriptomic analysis targeting apical radial glial cells (aRGCs), we found the downregulation of bRGC-related gene sets in patient-derived cerebral organoids. Lipidomic analysis revealed a decrease in the amount of lysophosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid, indicative of the disturbed phospholipid metabolism inPNPLA8knockout neural progenitor cells. Our data suggest that PNPLA8 has a critical role in the bRGC-mediated expansion of the developing human cortex by regulating the fate commitment of aRGCs.

Background: Pretreatment diagnosis of lateral pelvic lymph node (LPLN) metastasis is critical in deciding the therapeutic strategy for locally advanced rectal cancer. We aimed to investigate computed tomography (CT) cut-off values and diagnostic and prognostic implications of lateral pelvic lymph node metastasis (LPLNM) in rectal cancer. Methods: Patients with rectal cancer (n=126) who underwent primary tumor resection between April 2010 and July 2020 were included. CT findings and diagnostic accuracies were analyzed. Results: In this study, the cut-off values of 7 mm for long-axis diameter and 5 mm for short-axis diameter were selected. At cut-offs of 7 mm for long-axis diameter the values of sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 88.9%, 93.8%, 34.8%, 99.6%, and 93.7%, respectively. At cut-offs of 5 mm for short-axis diameter, the values of sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 88.9%, 90.9%, 26.7%, 99.5%, and 90.9%, respectively. CT findings of ≥7 mm for long-axis diameter (odds ratio, 68.0) and ≥5 mm for short-axis diameter (odds ratio, 38.4) were strong predictors of LPLNM. In subgroup analysis of patients with enlarged LPLN (≥7 mm), higher recurrence-free survival and lower local recurrence rates were found in patients who had LPLN dissection (5-year recurrence-free survival: 100% vs. 58.3%, p=0.17 with LPLN dissection; and 5-year local recurrence: 0% vs. 31.4%, p=0.27, without LPLN dissection). Conclusion: Pretreatment CT at cut-off values of 7 mm for long-axis diameter and 5 mm for short-axis diameter of LPLN provides sufficient diagnostic accuracy for LPLNM.

Supplementary Figures 1-7

Supplementary Tables 1-2

Supplementary Figure legends_R2

It is unknown as to how liver metastases are correlated with host immune status in colorectal cancer. In this study, we found that IL6, a proinflammatory cytokine produced in tumor-bearing states, promoted the metastatic colonization of colon cancer cells in association with dysfunctional antitumor immunity. In IL6-deficient mice, metastatic colonization of CT26 cells in the liver was reduced, and the antitumor effector function of CD8+ T cells, as well as IL12 production by CD11c+ dendritic cells, were augmented in vivo. IL6-deficient mice exhibited enhanced IFN-AR1–mediated type I interferon signaling, which upregulated PD-L1 and MHC class I expression on CT26 cells. In vivo injection of anti–PD-L1 effectively suppressed the metastatic colonization of CT26 cells in Il6−/− but not in Il6+/+ mice. Finally, we confirmed that colorectal cancer patients with low IL6 expression in their primary tumors showed prolonged disease-free survival. These findings suggest that IL6 may be a promising target for the treatment of metastasis in colorectal cancers by improving host immunity.

Supplementary Figure from Preoperative Chemoradiotherapy plus Nivolumab before Surgery in Patients with Microsatellite Stable and Microsatellite Instability–High Locally Advanced Rectal Cancer

Isolation of colon CICs as SP cells and the expression of OR7C1

Summary of genes overexpressed in SP cells

Purpose This study aimed to elucidate the influential predictive risk factors of Osgood–Schlatter disease (OSD) on the support (non-kicking) leg among adolescent soccer players considering peak height velocity (PHV) age and investigate the cut-off values of the predictive variables. Methods A cohort of 302 Japanese adolescent male soccer players aged 12–13 years were followed over 6 months. All players underwent physical examination, tibial tubercle ultrasonography, anthropometric and whole-body composition measurements, and muscle flexibility test of the support leg at the baseline. The developmental stage was evaluated from the PHV age. The OSD of the support leg was diagnosed 6 months later; players were divided into the OSD and control (CON) groups. The predictive risk factors were analyzed by multivariate logistic regression analysis. Results There were 42 players who had developed OSD at baseline and they were excluded from the study. Among the 209 players, 43 and 166 belonged to the OSD and CON groups, respectively. The predictive risk factors of OSD development were PHV age ± 6 months at baseline (p = 0.046), apophyseal stage of tibial tuberosity maturity at baseline (p p = 0.017), and decrease in gastrocnemius flexibility in 6 months (p = 0.009). Conclusion PHV age ± 6 months at baseline, apophyseal stage of the tibial tuberosity at baseline, quadriceps flexibility ≥ 35° at baseline, and decrease in gastrocnemius flexibility in 6 months are predictive risk factors of OSD development in the support leg among adolescent male soccer players. It is crucial to know the PHV age of each player, and not only the flexibility of quadriceps muscle but also the gastrocnemius should be monitored to predict OSD. Level of evidence II.

Supplemental Materials and Methods and figure legends

Summary of Colon cancer patients

OR7C1 gene overexpression experiments

Establishment of CIC-targeting immunotherapy using antigenic peptide derived from OR7C1.

Supplementary Data from Preoperative Chemoradiotherapy plus Nivolumab before Surgery in Patients with Microsatellite Stable and Microsatellite Instability–High Locally Advanced Rectal Cancer

Purpose: Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy.Experimental Design: Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model.Results: OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model.Conclusions: OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; 22(13); 3298–309. ©2016 AACR.

OR7C1 gene knockdown experiments

Summary of the OR7C1 peptides carrying HLA-A24 binding motief.

Purpose:Preoperative chemoradiotherapy (CRT) and surgical resection are the standard treatment for locally advanced rectal cancer (LARC). Combining immune checkpoint inhibitors with radiation suggests a promising approach for enhancing efficacy. We investigated the efficacy of CRT followed by nivolumab and surgery in patients with LARC.Patients and Methods:In phase I, we investigated the feasibility of sequentially combined CRT, 5 cycles of nivolumab, and radical surgery. In phase II, patients with microsatellite stable (MSS) and microsatellite instability-high (MSI-H) LARC were evaluated.Results:Three patients in phase I received full courses of CRT and nivolumab without dose modification; the schedule was recommended for phase II. A pathologic complete response (pCR) was centrally confirmed in 30% [11/37; 90% confidence interval (CI), 18%–44%] and 60% (3/5) of the MSS and exploratory MSI-H cohorts, respectively. While immune-related severe adverse events were observed in 3 patients, no treatment-related deaths were observed. In 38 patients with MSS who underwent surgery, pCR rates of 75% (6/8) and 17% (5/30; P = 0.004, Fisher exact test) were observed in those with programmed cell death ligand 1 (PD-L1) tumor proportion score ≥1% and P = 0.003, Fisher exact test) were observed for CD8+ T cell/effector regulatory T cell (CD8/eTreg) ratios of ≥2.5 and Conclusions:CRT followed by consolidation nivolumab could increase pCR. PD-L1 expression and an elevated CD8/eTreg ratio were positive predictors in patients with MSS LARC.

Background. Venoocclusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), is a life-threatening hematopoietic stem cell transplantation (HSCT) complication. Cases of mild and moderate VOD/SOS are self-limiting; however, the mortality for severe VOD/SOS has reached 80%. Recently, defibrotide became available and has been used for VOD/SOS; however, the outcome for patients with severe VOD/SOS is not satisfactory, and liver transplantation is attempted in these severe cases. Method. We describe a case of living donor liver transplantation (LDLT) for acute liver failure secondary to VOD/SOS that originates from HSCT. Result. Liver regeneration after LDLT was impaired, and several infections were developed before liver regeneration completion. Our patient suffered sepsis and finally died of multiorgan failure. Conclusion. Severe VOD/SOS originating from HSCT is associated with a very poor prognosis. The liver transplantation outcome for VOD/SOS has not been satisfied, but it may provide long-term survival if successful. We considered liver transplantation as a therapeutic option, especially in cases where sufficient graft volume is secured, considering impaired liver regeneration under bone marrow suppression after HSCT.