Supplementary Material for: Advanced Adenosquamous Carcinoma of the Ampulla of Vater Treated with Adjuvant Chemotherapy After Pancreaticoduodenectomy

Introduction: Adenosquamous carcinoma (ASC) of the ampulla of Vater (AmV) is rare. The prognosis is generally worse in patients undergoing resection of ASC of the AmV than in those undergoing resection of adenocarcinoma of the AmV because the former shows early recurrence after surgery. A treatment strategy for adenosquamous carcinoma of the AmV has not been established, and the efficacy of adjuvant chemotherapy after curative resection is unclear. Given the paucity of data, we report a case of ASC of the AmV that was curatively resected and treated with adjuvant chemotherapy. Case presentation: A 66-year-old man presented with pruritus and anorexia. Contrast-enhanced computed tomography revealed a tumor measusing 1.6 cm in diameter located at the AmV and distal bile duct . Biopsy revealed adenocarcinoma of the AmV. The patient underwent subtotal stomach-preserving pancreaticoduodenectomy. Histopathological examination contradictorily revealed adenosquamous carcinoma of the AmV and lymph node metastases. The postoperative course of the patient was uneventful, and he was discharged on day 25. The patient underwent S-1 adjuvant chemotherapy for 6 months and did not exhibit any postoperative recurrence for a follow-up duration of 28 months. Conclusion: Although treatment strategy for ASC of the AmV has not been established, our case shows that surgery followed by S-1 adjuvant chemotherapy could improve prognosis of patients with such tumors. However, further research is required to determine the efficacy of adjuvant chemotherapy and treatment strategies for resectable ASC of the AmV

Supplementary Material for: Effectiveness and Safety of Golimumab in the Treatment of Ulcerative Colitis: 52-week Results from Post-marketing Surveillance in Japan

Introduction: Real-world evidence for the effectiveness and safety of golimumab (GLM) in patients with ulcerative colitis (UC) is limited. The aim of this study was to investigate the 52-week effectiveness and safety of GLM treatment for UC. Methods: This prospective, multicentre, post-marketing surveillance study is conducted in 393 patients with UC in Japan (UMIN000027542). Clinical remission (partial Mayo score ≤2), adverse drug reactions (ADRs) and their predictors, and treatment persistence were analysed. Results: The safety analysis sets comprised 391 patients. Patients in clinical remission at baseline were excluded, and 336 were used for effectiveness analysis. Clinical remission was 47.9%, 48.5%, 44.6%, and 39.6% at weeks 6, 22, 36, and 52, respectively, in the intent-to-treat analysis. In biologic-naïve patients, clinical remission was slightly higher than that in biologic-experienced patients. At week 52, patients who concomitantly used corticosteroids at baseline showed numerically lower clinical remission rates than non-users of corticosteroids (34.9% vs 44.5%). Multivariate analysis showed that smoking history (P=0.040, odds ratio [OR]=1.911, 95% confidence interval [CI] 1.030–3.546) was an independent factor associated with clinical remission at week 52. ADRs occurred in 71 patients (18.2%) and included 9 cases of rash. Serious ADRs occurred in 40 patients (10.2%), including 8 cases of UC exacerbation. Additionally, the presence of comorbidities was associated with ADR incidence (P=0.010, OR=2.000, 95% CI 1.183–3.380). Conclusion: The real-world effectiveness of GLM treatment was confirmed in biologic-naïve and -experienced populations. The safety profile of GLM treatment was consistent with previous findings

Supplementary Material for: Chicken MDA5 Senses Short Double-Stranded RNA with Implications for Antiviral Response against Avian Influenza Viruses in Chicken

Mammalian melanoma differentiation-associated gene-5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) selectively sense double-stranded RNA (dsRNA) according to length, as well as various RNA viruses to induce an antiviral response. RIG-I, which plays a predominant role in the induction of antiviral responses against influenza virus infection, has been considered to be lacking in chicken, putting the function of chicken MDA5 (chMDA5) under the spotlight. Here, we show that chMDA5, unlike mammalian MDA5, preferentially senses shorter dsRNA synthetic analogues, poly(I:C), in chicken DF-1 fibroblasts. A requirement for caspase activation and recruitment domains for chMDA5-mediated chicken interferon beta (chIFNβ) induction and its interaction with mitochondrial antiviral signaling proteins were demonstrated. We also found that chMDA5 is involved in chIFNβ induction against avian influenza virus infection. Our findings imply that chMDA5 compensates in part the function of RIG-I in chicken, and highlights the importance of chMDA5 in the innate immune response in chicken

Supplementary Material for: Collectin Kidney 1 Plays an Important Role in Innate Immunity against Streptococcus pneumoniae Infection

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a <i>Streptococcus pneumoniae</i> infection analysis. First, we found that recombinant human CL-K1 bound to <i>S. pneumoniae</i> in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on <i>S. pneumoniae</i>. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on <i>S. pneumoniae</i> in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of <i>S. pneumoniae</i>. Consequently, less <i>S. pneumoniae</i> clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by<i> S. pneumoniae</i> infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against <i>S. pneumoniae</i> infection through the activation of the lectin complement pathway

Supplementary Material for: Favorable Impact of Serum TERT C228T for Prognosis after Surgical Resection for Liver Cancer

Introduction: Personalized medicine and molecular therapies with the diagnosis of somatic genetic alterations are expected to be developed for liver cancer. Nevertheless, it is unknown whether a mutation in the telomere reverse transcriptase promoter (TERT C228T) in serum cfDNA might be useful for making prognostic predictions after surgical resection for primary liver cancer. Methods: This cohort study retrospectively investigated 111 patients who had undergone surgical resection of liver cancer for the first time. We investigated the differences between clinicopathological features and prognosis according to classification of three tumor markers, including AFP, PIVKAII, and TERT C228T. Results: Multivariate analysis identified etiology (fatty liver disease vs. HBV odds ratio [OR] 6.853) and fibrosis stage (2–4, OR: 0.137) as determinants of TERT C228T-positive liver cancer with normal levels of AFP and PIVKAII (TERT single positive liver cancer). TERT single positive (Yes, OR: 0.301), fibrosis (FIB)-4 index (≥3.25, OR: 2.038), Child-Pugh classification (B, OR: 4.975), and number of tumors (≥2, OR: 4.098) were identified as determinants of the recurrence of liver cancer. TERT single positive (Yes, OR: 3.311), FIB-4 index (≥3.25, OR: 0.433), and number of tumors (≥2, OR: 0.262) were identified as determinants of disease-free survival. Conclusions: Our results highlight the impact of classification of prognostic tumor markers. TERT single positive is one predictor of favorable prognosis after surgical resection for liver cancer

Supplementary Material for: Efficacy and Safety of Oral Budesonide in Patients with Active Crohn's Disease in Japan: A Multicenter, Double-Blind, Randomized, Parallel-Group Phase 3 Study

<p><b><i>Background:</i></b> US and European guidelines recommend budesonide for the treatment of mild-to-moderate active ileocolic Crohn's disease (CD). However, budesonide has not been approved, and mesalazine is widely used as first-line treatment in Japan. The objective of this study was to evaluate the efficacy and safety of budesonide in patients with mild-to-moderate active CD in Japan. <b><i>Methods:</i></b> In this phase 3 noninferiority study (NCT01514240), 112 patients with a baseline Crohn's Disease Activity Index (CDAI) score of 180-400 were randomized to budesonide or mesalazine for 8 weeks. Assessments included remission rate (CDAI score ≤150) at weeks 2, 4, and 8, change in CDAI score, health-related quality of life (measured using the Inflammatory Bowel Disease Questionnaire [IBDQ]), and tolerability. <b><i>Results:</i></b> The remission rate at week 8 was numerically higher in the budesonide group (30.4%) than in the mesalazine group (25.0%), and the noninferiority of budesonide to mesalazine was shown. The mean total CDAI score decreased to a greater extent with budesonide than with mesalazine. Mean IBDQ scores improved from baseline to weeks 2, 4, 8, and 10 in both groups; improvements were numerically higher with budesonide than with mesalazine. No safety concerns were found. <b><i>Conclusion:</i></b> Budesonide is comparably effective to mesalazine in the treatment of Japanese patients with mild-to-moderate active CD.</p

Supplementary Material for: Prediction of Therapeutic Response Using Contrast-Enhanced Ultrasound in Japanese Patients Treated with Atezolizumab and Bevacizumab for Unresectable Hepatocellular Carcinoma

Introduction: Atezolizumab and bevacizumab (AB) therapy was the first-line treatment for unresectable hepatocellular carcinoma (u-HCC). However, the predictive marker of therapeutic response that can be easily used in clinical practice is still unknown. We prospectively investigated the utility of time-intensity curve (TIC) analysis using contrast-enhanced ultrasound (CEUS) as a predictive indicator of therapeutic response after the start of AB therapy. Methods: Thirty-five patients who received AB therapy for u-HCC were included in this study. TIC analysis was performed in 28 patients who were able to undergo CEUS before and 3–7 days after administration. We analyzed prognostic factors related to the initial therapeutic response and long progression-free survival (PFS). Results: The initial therapeutic response using dynamic computed tomography or Gd-EOB magnetic resonance imaging at 8–12 weeks after administration was partial response/stable disease/progressive disease (PD) in 14/12/9 cases (40/34/26%). Cases with PD (n = 9) had more cases without decreased blood flow in TIC analysis compared with cases with non-PD (100 vs. 18%, p = 0.001). Cases without decreased blood flow in TIC analysis (n = 10) had more cases with PD compared with cases with decreased blood flow (60 vs. 0%, p = 0.001). PFS in patients without decreased blood flow early after the administration was shorter than that in those with decreased blood flow (9.1 vs. 28 weeks, p = 0.0051). Conclusion: Early evaluation by TIC analysis using CEUS may be useful in predicting the therapeutic response in patients treated with AB therapy for u-HCC

Supplementary Material for: Cortical Actin Alteration at the Matrix-Side Cytoplasm in Lung Adenocarcinoma Cells and Its Significance in Invasion

<p><b><i>Objectives:</i></b> Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas. <b><i>Materials and Methods:</i></b> The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells. <b><i>Results:</i></b> In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP. <b><i>Conclusion:</i></b> Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.</p

Supplementary Material for: Efficacy and Safety of Dose Escalation to Adalimumab 80 mg Every Other Week in Japanese Patients with Crohn Disease Who Lost Response to Maintenance Therapy

<b><i>Background:</i></b> Dose escalation is often recommended for loss of response in anti-TNFα-treated patients with Crohn disease (CD). This 52-week phase 3, multicenter study investigated the efficacy and safety of escalation to adalimumab 80 mg every other week (EOW) in Japanese patients with CD who lost response to maintenance adalimumab 40 mg EOW. <b><i>Methods:</i></b> Twenty-eight patients aged ≥15 years with moderately to severely active CD who had previously attained and subsequently lost clinical response to maintenance adalimumab received open-label adalimumab 80 mg EOW during weeks 0–50. Loss of response was defined as CD activity index (CDAI) ≥200, increases in CDAI ≥50 from minimum observed value, and C-reactive protein (CRP) ≥1 mg/dL at screening. The primary endpoint was the proportion of patients achieving a CDAI decrease ≥50 (CR-50) from baseline at week 8. <b><i>Results:</i></b> At weeks 8 and 52, 75.0 and 57.1% of patients achieved CR-50 and 25.0 and 35.7% achieved clinical remission (CDAI < 150), respectively; median CRP changes from baseline were −0.39 and −0.77 mg/dL, respectively. Most treatment-emergent adverse events were mild to moderate. <b><i>Conclusions:</i></b> Adalimumab dose escalation to 80 mg EOW improved CD activity in patients who had lost response to maintenance adalimumab, with no new safety signals

Supplementary Material for: Association of Cancer and Its Interaction with Conventional Risk Factors on Cardiovascular Disease Risk

Introduction We sought to examine the association of cancer history with the incidence of individual cardiovascular disease events and to clarify whether the history of cancer modifies the relationship between conventional cardiovascular risk factors and incident cardiovascular disease. Methods This retrospective cohort study used the JMDC Claims Database, including 3,531,683 individuals. The primary endpoint was the composite cardiovascular disease outcome, which included myocardial infarction, angina pectoris, stroke, heart failure, and atrial fibrillation. Results During a follow-up, 144,162 composite endpoints were recorded. Individuals with a history of cancer had a higher risk of developing composite cardiovascular disease events (HR 1.26, 95% CI 1.22-1.29). The HRs for myocardial infarction, angina pectoris, stroke, heart failure, and atrial fibrillation were 1.11 (95% CI 0.98-1.27), 1.15 (95% CI 1.10-1.20), 1.11 (95% CI 1.05-1.18), 1.39 (95% CI 1.34-1.44), and 1.22 (95% CI 1.13-1.32), respectively. Individuals who required chemotherapy for cancer had a higher risk of developing cardiovascular disease. Although conventional risk factors (e.g., overweight/obesity, hypertension, and diabetes) were associated with incident composite cardiovascular disease even in individuals with a history of cancer, the total population-attributable fractions of conventional risk factors were less in individuals with a history of cancer. Conclusion Individuals with a history of cancer (particularly those requiring chemotherapy) have a higher risk of cardiovascular disease. Traditional risk factors are important in the development of cardiovascular disease in individuals with and without a history of cancer. In individuals with a history of cancer, however, the total population-attributable fractions of conventional risk factors decreased