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We present the case of an elderly female patient with rheumatoid arthritis (RA) treated with methotrexate. She was referred to our hospital with severe malaise. She was emaciated and had massive pleural effusion that induced atelectasis. Her blood tests revealed elevated CRP, leukopenia, and severe anaemia. She lost consciousness on the third day of hospital stay and passed away the following day. Her autopsy showed gelatinous transformation of the bone marrow that gave rise to bicytopoenia, whereas there were no other causes for severe anaemia. Bone marrow gelatinous transformation can cause impaired haematopoiesis in elderly RA patients.

Introduction Recently, nephronophthisis (NPH) has been considered a monogenic cause of end-stage renal disease (ESRD) in adults. However, adult-onset NPH is difficult to accurately diagnose and has not been reported in a cohort study. In this study, we assessed the genetic background and clinicopathologic features of adult NPH. Methods We investigated 18 sporadic adult patients who were suspected as having NPH by renal biopsy. We analyzed 69 genes that cause hereditary cystic kidney disease and compared clinicopathologic findings between patients with and without pathogenic mutations in NPH-causing genes. Results Seven of 18 patients had pathogenic NPH-causing mutations in NPHP1, NPHP3, NPHP4, or CEP164. Compared with patients without pathogenic mutations, those with pathogenic mutations were significantly younger but did not significantly differ in the classic NPH pathologic findings, such as tubular cysts. On the other hand, the number of tubules with thick tubular basement membrane (TBM) duplication, which was defined as >10-μm thickness, was significantly higher in patients with genetically proven adult NPH than in those without pathogenic mutations. α-Smooth muscle actin (α-SMA)-positive myofibroblasts were detected inside thick TBM duplication. Conclusions In adult patients with NPH, thick TBM duplication was the specific finding. Our analysis also suggested that older patients tended to have no pathogenic mutations, even when they were suspected to have NPH by renal biopsy. These findings could be the novel clinical clue for the diagnosis of NPH in adult patients., Graphical abstract

Background We previously reported that PD-L1 polymorphisms are associated with the efficacy and immune-related adverse events of PD-1 blockade with nivolumab. However, the association between PD-L1 polymorphisms and survival outcomes under PD-1/PD-L1 blockade is still uncertain. Here, we aimed to investigate whether PD-L1 polymorphisms are associated with survival outcomes in advanced non-small-cell lung cancer (NSCLC) patients treated with nivolumab. Methods PD-1/PD-L1 polymorphisms and survival outcomes were retrospectively analysed in two independent cohorts (133 patients treated with nivolumab and 96 patients with no treatment history of an immune checkpoint inhibitor (ICI) (the non-ICI cohort)) with advanced NSCLC. Results Among the 7 studied single-nucleotide polymorphisms, PD-L1 rs822339 and rs1411262 were associated with overall survival (OS) in patients treated with nivolumab. Patients with the A/A genotype of rs822339 had a significantly longer OS than those with A/G or G/G genotypes (not reached versus 12.0 months; hazard ratio (HR), 0.35; 95% confidence interval (CI), 0.18–0.64; p = 0.0008). A similar survival benefit with the A/A genotype was observed regardless of driver mutation status. In multivariate analysis, performance status (PS) and PD-L1 rs822339 genotype were independent prognostic factors for OS. In the non-ICI cohort, the PD-L1 rs822339 genotype did not correlate with OS (HR, 0.77; 95% CI, 0.31–1.70; p = 0.55). The T/T genotype of rs1411262 also showed a significant prolongation of OS compared to that with the C/T or C/C genotypes in patients treated with nivolumab. Conclusions PD-L1 polymorphisms are associated with favourable OS in nivolumab-treated NSCLC patients and may be useful predictive biomarkers, regardless of driver mutation status.

Objective To assess the utility of preoperative magnetic resonance imaging (MRI) in the diagnosis of tubal pregnancy. Summary of background data Most cases of ectopic pregnancy are tubal pregnancies. Preoperative accurate diagnosis including the location of the tubal pregnancy is important. Method We performed a retrospective single-center cohort study evaluating patients who underwent surgery for ectopic pregnancy from April 2004 to March 2016 and who underwent preoperative MRI. Sixty patients were enrolled in the study. Chorion confirmed at the tubal pregnancy site detected by MRI was defined as a correct diagnosis. Results Mean estimated gestational age at the time of MRI according to the last menstrual period was 7 weeks (median: 5 weeks; range: 3–10 weeks). Ectopic pregnancy was diagnosed by MRI in 57 cases, and all cases were either left or right tubal pregnancy. In 52 cases, the actual position matched the MRI findings. In 5 cases, the location of tubal ectopic pregnancy was different from that diagnosed by MRI, or no tubal pregnancy was observed intraoperatively. In 2 cases, no ectopic pregnancy site was observed on MRI, but tubal pregnancy was confirmed by surgery. In one case, an ectopic pregnancy site was not found, and the tube showed normal findings on MRI. The sensitivity of MRI diagnosis was 96.2%, specificity was 16.7%, positive predictive value was 91.1%, and negative predictive value was 33.3%. Conclusion In cases of ectopic pregnancy treated surgically, the sensitivity of MRI diagnosis, including identification of the location of ectopic pregnancy, is high.

The process of diagnostic errors among Japanese residents has not been previously studied. This descriptive study was conducted in June 2019 on junior residents at a single‐center educational hospital in Japan. Diagnosis Error Evaluation and Research taxonomy was used to measure the process of diagnostic error in the most memorable error cases. High frequency of diagnostic errors resulted from inaccurate/misinterpretation of history, failure/delay in eliciting physical examination findings, inaccurate/misinterpretation of physical examination, failure in weighting of physical examination, and failure/delay in considering the diagnosis. Residents made diagnostic errors mainly during history taking, physical examination, and assessment., According to the analysis of diagnostic error cases among Japanese residents using Diagnosis Error Evaluation and Research taxonomy, diagnostic errors are mainly caused during medical history collection, physical examination, and clinician evaluation.

Resistant hypertension is an important cardiovascular risk factor. This analysis of the JAMP study (Japan Ambulatory Blood Pressure Monitoring Prospective) data investigated the effects of uncontrolled resistant hypertension diagnosed using ambulatory blood pressure (BP) monitoring on the risk of heart failure (HF) and overall cardiovascular events. The JAMP study patients with hypertension and no HF history were included. They had true resistant hypertension (24-hour BP ≥130/80 mm Hg), pseudoresistant hypertension (24-hour BP P =0.012 and 2.24 [95% CI, 1.17–4.30]; P =0.015, respectively) and versus uncontrolled nonresistant hypertension (1.51 [1.03–2.20]; P =0.034 and 3.03 [1.58–5.83]; P Registration: URL: https://www.umin.ac.jp/ctr ; Unique identifier: UMIN000020377.

Lower gastrointestinal perforation is rare and challenging to diagnose timeously in the acute abdomen. However, no study has examined the frequency and associated factors of diagnostic errors related to lower gastrointestinal perforation. This large-scale multicenter retrospective study investigated the frequency of diagnostic errors and identified the associated factors. This study retrospectively analyzed data on patient factors, symptoms, situational factors, and physician factors across nine institutions between January 1, 2015 and December 31, 2019. Timely diagnosis was defined as diagnosis at the first visit to a computed tomography (CT)-capable facility or referral to an appropriate medical institution immediately following the first visit to a non-CT-capable facility; cases not meeting this definition were defined as diagnostic errors that resulted in delayed diagnosis. Of the 439 cases of lower gastrointestinal perforation identified, delayed diagnosis occurred in 138 cases (31.4%). Multivariate logistic regression analysis showed a significant association between examination by a non-generalist and delayed diagnosis. Other factors, such as presence of fever, absence of abdominal tenderness, and unavailability of urgent radiology reports, tended to be associated with delayed diagnosis. The initial misdiagnoses mainly comprised gastroenteritis, constipation, and small bowel obstruction. In conclusion, diagnostic errors occurred in about one-third of lower gastrointestinal perforation cases.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection., Graphical Abstract, NOD-SCID mice orally inoculated with 9 × 109 colony-forming units of Shiga toxin-producing Escherichia coli (STEC) O111 and treated 48 h later with an intravenous injection of 5 × 104 human bone marrow-derived multilineage-differentiating stress-enduring (Muse) cells exhibited 100% survival. Thus, the intravenous administration of Muse cells might be a candidate therapeutic approach for preventing acute encephalopathy after STEC infection.

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer., Relapse is a limitation for the efficacy of the anaplastic lymphoma kinase (ALK)-inhibitor alectinib in ALK-rearranged lung cancer. Here, the authors show that YAP1 activation upon alectinib treatment leads to therapy resistance and that inhibiting both YAP1 and ALK leads to longer tumor remission in mice.

Giant cell arteritis (GCA) is a type of vasculitis that occurs among the elderly and is categorised as granulomatous vasculitis of large- and medium-sized vessels. We herein report a case of GCA in a 78-year-old woman with an 11-month history of erythema nodosum (EN). She presented with fever, chest pain and headache. Inflammatory markers, including C-reactive protein and the erythrocyte sedimentation rate, were elevated. Computed tomography (CT) revealed thickening of the arterial walls from the aortic arch. Positron emission tomography/CT showed uptake of 18F-fluorodeoxyglucose in the walls of the proximal left common carotid and left subclavian arteries. The presence of temporal arteritis could not be confirmed. We diagnosed the patient with large-vessel GCA (LV-GCA). Induction therapy with prednisolone resulted in the rapid amelioration of her symptoms and inflammation. Cutaneous manifestations other than scalp necrosis in GCA are uncommon. In this case, EN preceded the onset of LV-GCA. The present case suggests EN can be a clinical manifestation of LV-GCA.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a recently identified target antigen of idiopathic membranous nephropathy (iMN). The clinicopathological characteristics of THSD7A-associated MN are poorly characterised due to low prevalence among MN patients. Among 469 consecutive cases of pathologically confirmed MN diagnosed at four centres in Japan, 14 cases were confirmed positive for THSD7A by immunohistochemistry (3.0%). The prevalence of THSD7A-associated MN tended to be higher in northern Japan. Most cases demonstrated nephrotic-range proteinuria (12/14 cases, 86%). In two patients, cancer was detected at the time of renal biopsy (small-cell carcinoma of the lung and prostatic adenocarcinoma with neuroendocrine differentiation). Both tumours were negative for THSD7A. Four patients had concurrent or previous incidence of allergic diseases, including one patient with Kimura’s disease. Pathological analysis of kidney biopsy tissue revealed slight mesangial cell proliferation in three cases and spike formation in one case. Immunofluorescence studies demonstrated that IgG subclass was mainly IgG4-dominant/codominant (12/13, 92% cases), while the case with prostatic cancer had an IgG2-dominant distribution. The immunostaining profile for components of the lectin complement pathways was not significant in three cases including two patients with malignancy. One case was dual positive for THSD7A and PLA2R. Of 10 cases with known clinical follow-up data, 6 demonstrated reduced serum creatinine and 8 presented reduced proteinuria. In summary, although the major IgG phenotype was usually IgG4-dominant/codominant, clinical background was otherwise heterogeneous. Further investigation of regional differences in THSD7A-associated MN prevalence may reveal genetic and environmental risk factor and associated pathogenic mechanisms. Electronic supplementary material The online version of this article (10.1007/s00428-019-02558-0) contains supplementary material, which is available to authorized users.

An 83-year-old man was transported by ambulance to the emergency room because he had a clouding of consciousness. On arrival, his Glasgow Coma Scale score was 13 (E4V4M5). His temperature was 37.3°C, blood pressure 139/77 mm Hg and pulse 101 beats per minute. Physical examination revealed jaundice and purpura throughout the body. Laboratory evaluation revealed a haemoglobin level of 5.7 g/dL, thrombocytopenia (platelet count: 0.6×104/μL), …

Summary A 31-year-old man with Williams syndrome (WS) was referred to our hospital because of a 9-year history of hypertension, hypokalemia, and high plasma aldosterone concentration to renin activity ratio. A diagnosis of primary aldosteronism (PA) was clinically confirmed but an abdominal CT scan showed no abnormal findings in his adrenal glands. However, a 13-mm hypervascular tumor in the posterosuperior segment of the right hepatic lobe was detected. Adrenal venous sampling (AVS) subsequently revealed the presence of an extended tributary of the right adrenal vein to the liver surrounding the tumor. Segmental AVS further demonstrated a high plasma aldosterone concentration (PAC) in the right superior tributary vein draining the tumor. Laparoscopic partial hepatectomy was performed. The resected tumor histologically separated from the liver was composed of clear cells, immunohistochemically positive for aldesterone synthase (CYP11B2), and subsequently diagnosed as aldosterone-producing adrenal adenoma. After surgery, his blood pressure, serum potassium level, plasma renin activity and PAC were normalized. To the best of our knowledge, this is the first report of WS associated with PA. WS harbors a high prevalence of hypertension and therefore PA should be considered when managing the patients with WS and hypertension. In this case, the CT findings alone could not differentiate the adrenal rest tumor. Our case, therefore, highlights the usefulness of segmental AVS to distinguish adrenal tumors from hepatic adrenal rest tumors. Learning points: Williams syndrome (WS) is a rare genetic disorder, characterized by a constellation of medical and cognitive findings, with a hallmark feature of generalized arteriopathy presenting as stenoses of elastic arteries and hypertension. WS is a disease with a high frequency of hypertension but the renin-aldosterone system in WS cases has not been studied at all. If a patient with WS had hypertension and severe hypokalemia, low PRA and high ARR, the coexistence of primary aldosteronism (PA) should be considered. Adrenal rest tumors are thought to arise from aberrant adrenal tissues and are a rare cause of PA. Hepatic adrenal rest tumor (HART) should be considered in the differential diagnosis when detecting a mass in the right hepatic lobe. Segmental adrenal venous sampling could contribute to distinguish adrenal tumors from HART.

Autism spectrum disorder (ASD) occurs in 1 in 160 children worldwide. Individuals with ASD tend to be unique in the way that they comprehend themselves and others, as well as in the way that they interact and socialize, which can lead to challenges with social adaptation. There is currently no medication to improve the social deficit of children with ASD, and consequently, behavioral and complementary/alternative intervention plays an important role. In the present pilot study, we focused on the neuroendocrinological response to participatory art activities, which are known to have a positive effect on emotion, self-expression, sociability, and physical wellbeing. We collected saliva from 12 children with ASD and eight typically developed (TD) children before and after a visual art-based participatory art workshop to measure the levels of oxytocin, a neuropeptide involved in a wide range of social behaviors. We demonstrated that the rate of increase in salivary oxytocin following art activities in ASD children was significantly higher than that in TD children. In contrast, the change rate of salivary cortisol after participatory art activities was similar between the two groups. These results suggest that the beneficial effects of participatory art activities may be partially mediated by oxytocin release, and may have therapeutic potential for disorders involving social dysfunction.

In the central nervous system, many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. In this study, we investigated the regulation of spatiotemporal group dynamics of stationary mitochondria. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in vitro and in vivo. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of neighboring mitochondria. Furthermore, we showed that the ATP concentration was relatively high around mitochondria, and treating axons with phosphocreatine, which supplies ATP, reduced the immobile mitochondria induced by local mitochondrial inhibition. These observations indicate that intermitochondrial interactions, mediated by ATP signaling, control the uniform distribution of axonal mitochondria. The present study reveals a novel cellular system that collectively regulates stationary mitochondria in axons.

Aim: Chronic active antibody-mediated rejection (CAABMR) is an important cause of late-stage renal allograft loss. Early inflammatory events such as acute rejection and infection after transplantation are considered to be the risk factors of de novo donor-specific antibody (dnDSA) production. In this study, we investigated the relationship between pre­disposing T-cell-mediated rejection and dnDSA-positive CAABMR. Methods: We recruited 365 patients who underwent ABO-compatible renal transplantation at our hospital. Among them, 16 patients diagnosed as having dnDSA-positive CAABMR were designated as a CAABMR group, and 38 randomly selected patients were designated as a control group. All biopsies from 1 month after transplantation were included in the study. The presence or absence of borderline changes (BLCs), acute T-cell-mediated rejection (ATMR), microvascular inflammation (MVI), and C4d positive on peritubular capillaries (C4d-P) was examined. Results: In the CAABMR group, BLC/ATMR was found in 12 cases (75%), and the mean duration until appearance of BLC/ATMR was 282.7 ± 328.7 days. C4d-P was found in 11 cases (68.8%), and the mean duration until its appearance was 1,432 ± 1,307 days. MVI was found in all cases, and the mean duration until its appearance was 1,333 ± 1,126 days. The mean duration until diagnosis of CAABMR was 2,268 ± 1,191 days. In the control group, BLC/ATMR was found in 13 cases (34.2%), and the mean duration until the appearance of BLC/ATMR was 173.1 ± 170.4 days. C4d-P was found in 2 cases (5.3%), and the durations until its appearance were 748 and 1,881 days. No cases of MVI were found in the control group. The frequency of BLC/ATMR was significantly higher in the CAABMR group (p < 0.01). Conclusion: Preceding BLC/ATMR is associated with the development of CAABMR with dnDSA.

The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity in vitro (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size in vivo. To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients. Implications: This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

Patients with autoimmune diseases (AIDs) may develop lymphoproliferative disorders (LPDs) during treatment with immunosuppressive agents (IS) such as methotrexate (MTX), biological agents, or tacrolimus. Some LPDs in patients with AIDs (AID-LPDs) regress after withdrawal of IS, and a high incidence of Epstein-Barr virus (EBV) positivity in such patients has been reported. To identify characteristics and factors predictive of the response to treatment and disease progression, we retrospectively analyzed clinical and histopathological data for 81 patients with AID-LPDs. Almost all of them (96%) had been treated with MTX. Diffuse large B cell lymphoma was the most common LPD type (61%) and seven patients (9%) had classical Hodgkin lymphoma (CHL). EBV was detected by in situ hybridization with an EBV-encoded small RNA (EBER) probe in 43% of the examined cases. In 59 patients, IS was discontinued as the initial treatment, resulting in regression of LPDs in 69% of them, and multivariate analysis showed that EBER positivity was an independent factor predictive of such regression (p = 0.022). Two-year progression-free survival (PFS) and overall survival for the patients overall were 63% and 83%, respectively. Poor PFS was associated with advanced stage (p = 0.024), worse performance status (PS, p = 0.031), CHL histology (p = 0.013), and reactivation of EBV-related antibodies (p = 0.029). In conclusion, EBV positivity demonstrated using an EBER probe is useful for prediction of successful regression after withdrawal of IS in patients with AID-LPDs. Patients with advanced stage disease, worse PS, CHL histology, or reactivation of EBV-related antibodies should be closely monitored after initial treatment.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder estimated by the World Health Organization to occur in one of 160 children worldwide. No pharmaceutical treatments are available to improve the deficits in social communication that are common symptoms of ASD. Recent clinical trials have focused on the nasal application of oxytocin, a neuronal peptide known to regulate a variety of social behaviours. However, the effect of oxytocin on this deficit is inconclusive. By contrast, evidence from ASD animal model studies indicates that when animals are treated with oxytocin during early development, improvements in social deficits are observed in adulthood. Thus, it is necessary to examine the effect of therapeutic target medication prescribed in early development. Mice prenatally exposed to valproic acid (VPA) are widely used as an animal model of ASD. However, many behavioural studies have been conducted during adulthood rather than early development. To establish a screening system to identify therapeutic drugs that are effective when delivered during the early postnatal period, it is important to examine the early developmental changes in their communicative behaviours. In the present study, we examined the ultrasonic vocalisation (USV) of VPA-exposed mice pups during their early postnatal developmental days. USV rates were comparable to those of the controls until the first week of their life but declined more on postnatal day 11. We checked the expression of oxytocin system in the hypothalamus and found the down-regulation of oxytocin and CD38, and up-regulation of oxytocin receptor in the VPA pups. Acute administration of oxytocin on postnatal day 11 increased the call rate of VPA pups. Taken together, we have demonstrated there was a deficiency in the oxytocinergic signalling in the VPA pups and also shown the existence of time periods that are effective with respect to screening the therapeutic drugs.

There have been few advances in the treatment of small-cell lung cancer (SCLC) because of the lack of targets. MCL1, a member of the anti-apoptotic BCL-2 family, may be a treatment target in several cancers, including SCLC. However, whether the expression profile of the anti-apoptotic BCL-2 family affects MCL1 inhibition strategy is unknown. A tissue microarray (TMA) was created from consecutive patients who were diagnosed with SCLC and had previously undergone surgery at Kyoto University Hospital (Kyoto, Japan) between 2001 and 2017. We used S63845, a MCL1 inhibitor, to assess the cytotoxic capacity in SCLC cell lines including a patient-derived cell line in vitro and in vivo. The combination of S63845 with navitoclax, a double BCL-XL/BCL-2 inhibitor, was also employed to examine the comprehensive inhibition of the anti-apoptotic BCL-2 family. Immunohistochemistry of a TMA from patients with surgically resected SCLC demonstrated high MCL1 expression with low BCL-XL and BCL-2 to be the most common expression profile. S63845 was effective in high MCL1- and low BCL-XL-expressing SCLC cell lines. S63845 induced BAK-dependent apoptosis in vitro, and the anti-tumor efficacy was confirmed in an in vivo model. Although knockdown of BCL-XL and BCL-2 improved the cytotoxic activity of S63845 and its combination with navitoclax increased the anti-tumor cytotoxicity, the therapeutic range of S63845 with navitoclax was narrow in in vivo studies. Our study suggests MCL1 inhibition therapy be applied for high MCL1- and low BCL-XL-expressing SCLC patients.

// Taku Murakami 1 , Cindy M. Yamamoto 1 , Tomoshige Akino 2 , Hiroshi Tanaka 2 , Nobuyuki Fukuzawa 2 , Hidetaka Suzuki 2 , Takahiro Osawa 3 , Takahiro Tsuji 2 , Toshimori Seki 2 and Hiroshi Harada 2 1 Hitachi Chemical Co. America, Ltd., Irvine, CA, USA 2 Sapporo City General Hospital, Sapporo, Japan 3 Graduate School of Medicine, Hokkaido University, Sapporo, Japan Correspondence to: Taku Murakami, email: tmurakami@hitachi-chemical.com Keywords: bladder cancer; biomarker; extracellular vesicles; exosome; mRNA Received: May 03, 2018 Accepted: August 04, 2018 Published: August 28, 2018 ABSTRACT Objective: Urinary extracellular vesicles (EV) could be promising biomarkers for urological diseases. In this retrospective feasibility study, we conducted biomarker screening for early stage bladder cancer using EV mRNA analysis. Methods: Biomarker candidates were identified through RNA-seq analysis of urinary EV from patients with non-muscle invasive bladder cancer (N=3), advanced urothelial cancer (N=3), no residual tumor after TURBT (N=2), and healthy and disease controls (N=4). Diagnostic performance was evaluated by RT-qPCR in a larger patient group including bladder cancer (N=173), renal pelvis and ureter cancer (N=33), no residual tumor and non-cancer disease control (N=36). Results: Urinary EV SLC2A1 , GPRC5A and KRT17 were overexpressed in pT1 and higher stage bladder cancer by 20.6-fold, 18.2-fold and 29.5-fold, respectively. These genes allowed detection of non-muscle invasive bladder cancer (AUC: 0.56 to 0.64 for pTa, 0.62 to 0.80 for pTis, and 0.82 to 0.86 for pT1) as well as pT2 and higher muscle invasive bladder cancer (AUC: 0.72 to 0.90). Subgroup analysis indicated that these markers could be useful for the detection of cytology-negative/-suspicious and recurrent bladder cancers. Conclusion: Three urinary EV mRNA were discovered to be elevated in bladder cancer. Urinary EV mRNA are promising biomarkers of urothelial cancer and worth further investigation.

Background: Alectinib is a new standard treatment for treatment-naïve anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC); however, resistance ultimately develops in almost all patients, and data regarding the efficiency of ceritinib for such patients are insufficient. Patients and Methods: Patients with ALK-positive NSCLC treated at the Kyoto University Hospital from January 2012 to March 2017 were reviewed. Patients who were treated with ceritinib after alectinib were identified, and the efficacy of ceritinib after alectinib was retrospectively evaluated. Results: There were 35 patients with ALK-positive NSCLC, nine of whom received ceritinib after alectinib. The overall response rate to ceritinib was 44%. It was 16% in patients who received ceritinib immediately after alectinib, and 100% in patients who received chemotherapy before ceritinib. The median progression-free survival for patients treated with ceritinib was 4.4 months (95% confidence interval(CI)=1.1-6.5 months). Conclusion: Ceritinib demonstrated a modest clinical benefit after failure of alectinib. Ceritinib may be a reasonable treatment option in this setting.

Bleeding is the most important clinical outcome in patients with immune thrombocytopenia (ITP), and the goal of therapy in such cases is to treat or prevent bleeding. The frequency of and risk factors for bleeding events in ITP have only recently been identified in several large-scale studies. However, there is little published information about severe life-threatening bleeding in ITP. To clarify the clinical features of life-threatening bleeding in patients with primary ITP, we systematically reviewed the medical records of all ITP patients that were admitted to our hospital between January 1, 1992, and December 31, 2015. Of 169 consecutive inpatients with primary ITP, 8 suffered life-threatening bleeding (10 episodes: gastrointestinal, 4 cases; pulmonary, 1 case; and intracranial, 5 cases). All of these patients were ≥ 60 years of age and had platelet counts of

Lung cancer with preexisting interstitial lung disease (ILD) is difficult to treat due to the risk of acute exacerbation of ILD. Nanoparticle albumin-bound (nab-) paclitaxel improves the overall response rate and reduces neuropathy more efficiently compared with conventional solvent-based (sb-) paclitaxel in patients with advanced non-small-cell lung cancer. However, it is not known whether the risk of acute exacerbation of ILD with nab-paclitaxel is higher compared with that with sb-paclitaxel. Advanced lung cancer patients with ILD treated with nab-paclitaxel (n=14) or sb-paclitaxel (n=14) were retrospectively reviewed. Acute exacerbation of ILD developed in 1/14 patients (7.7%) receiving nab-paclitaxel and 3/14 patients (21.4%) receiving sb-paclitaxel; the difference was not statistically significant. To the best of our knowledge, this is the first study to compare the incidence of acute exacerbation of ILD with nab-paclitaxel with that of sb-paclitaxel in patients with advanced lung cancer with preexisting ILD. The results of the present study support conducting a prospective clinical trial to confirm the clinical benefit of this agent.

Key points A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light-induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo-entrainment of biological rhythms. Abstract In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light-evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c-fos in the SCN, which is involved in photic entrainment of circadian rhythms.

AIM To evaluate the utility of plasma circulating tumor DNA (ctDNA) using the peptide nucleic acid-locked nucleic acid (PNA-LNA) clamp method to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients who had progressed under treatment with EGFR-tyrosine kinase inhibitors (TKIs). PATIENTS AND METHODS Blood samples were collected from patients with EGFR mutation-positive NSCLC who had progressed on EGFR-TKIs between March 2016 and August 2016 at the Kyoto University Hospital. Extracted ctDNA was analyzed using the PNA-LNA clamp method. In eligible patients, tissue re-biopsy was also performed and EGFR mutation status was compared between tissue and plasma samples. RESULTS Thirty-one patients were enrolled in this study. Known activating EGFR mutations and the T790M mutation were detected in 18 (58%) and 5 patients (16%), respectively. Twenty-five patients underwent tissue re-biopsy. Adequate samples for mutation analysis were obtained from 21 patients and 10 patients were found to be tissue T790M-positive. Among these 10 patients, 4 patients were positive for T790M in plasma ctDNA (sensitivity 40% and specificity 100%). All patients with T790M-positive plasma ctDNA responded to osimertinib. CONCLUSION Sensitivity of the PNA-LNA clamp method in detecting the plasma EGFR T790M mutation was moderate with elevated, however, specificity. Plasma EGFR T790M testing may be adequate for the initial step; however, tissue re-biopsy should be considered for plasma T790M-negative patients because of its high false-negative rate.

Odorant molecules stimulate olfactory receptor neurons, and axons of these neurons project into the main olfactory bulb where they synapse onto mitral and tufted cells. These project to the primary olfactory cortex including the anterior olfactory nucleus (AON), the piriform cortex, amygdala, and the entorhinal cortex. The properties of mitral cells have been investigated extensively, but how odor information is processed in subsequent brain regions is less well known. In the present study, we recorded the electrical activity of AON neurons in anesthetized rats. Most AON cells fired in bursts of 2–10 spikes separated by very short intervals (, The output cells of the main olfactory bulb project via the lateral olfactory tract (LOT) to the anterior olfactory nucleus (AON), but how AON neurons process that information is not well understood. We recorded the electrical activity of AON neurons in anaesthetized rats, and studied their responses to diverse odors and to electrical stimulation of the LOT. Most AON cells fired in bursts linked to the respiratory rhythm. The rhythms of adjacent cells, while locked to the same underlying rhythm, often showed marked differences in phase. Some AON cells showed short latency excitation following stimulation of the LOT, indicating that they were primary recipients of afferents from the olfactory bulb; these all fired burst during inspiration. However, most AON cells were inhibited by LOT stimulation, indicating that they received inhibitory projections from the primary recipients, and most of these cells fired bursts during expiration. We discuss the implications of these observations for the coding of odor information.

Nivolumab improves the prognosis of non-small cell lung cancer (NSCLC) but can cause immune-related adverse events (irAEs). Reports have indicated longer progression-free survivals (PFSs) in patients with irAEs than in those without irAEs. We reported associations between programmed death ligand-1 (PD-L1) single nucleotide polymorphisms (SNPs) and PFS after nivolumab treatment. We hypothesized that adverse events might be associated with the SNPs of PD-L1. We analyzed data from 111 patients with NSCLC treated with nivolumab. The response rate was 14%, and the median PFS was 68 days. We found patients with the adverse event of low free tetraiodothyronine (fT4) had significantly longer PFSs than those without low fT4 (not reached vs 67 days; hazard ratio [HR], 0.297; P = 0.010). Moreover, median overall survival was longer in patients with low fT4 than those without low fT4 (not reached vs 556 days, HR, 0.139; P = 0.020). Patients with the T allele of rs1411262 (P = 0.0073) and with the A allele of rs822339 (P = 0.0204) developed low fT4. Patients with the T/T genotype had longer PFSs than with those with the C/T and C/C genotypes of rs1411262 (165 vs. 67 days, HR, 1.65; P = 0.040), and those with the A/A genotype had longer PFSs than those with the A/G and G/G genotypes of rs822339 (182 vs. 67 days, HR, 1.76; P = 0.025). In the patients with advanced NSCLC, low fT4 after nivolumab treatment was associated with significantly longer PFSs. The SNPs of PD-L1 may be associated with the adverse events of nivolumab.

Chronic active antibody-mediated rejection (CAAMR) is a particular problem in kidney transplantation (KTx), and ~25% of grafts are lost by CAAMR. Further, the pathogenesis remains unclear, and there is no effective cure or marker. We previously found that a hyper NFκB-activating mechanism in non-immune cells, called the IL-6 amplifier, is induced by the co-activation of NFκB and STAT3, and that this activation can develop various chronic inflammatory diseases. Here, we show that synaptotagmin-17 (SYT17) is increased in an exosomal fraction of the urine from CAAMR patients, and that this increase is associated with activation of the IL-6 amplifier. Immunohistochemistry showed that SYT17 protein expression was increased in renal tubule cells of the CAAMR group. While SYT17 protein was not detectable in whole-urine samples by western blotting, urinary exosomal SYT17 levels were significantly elevated in the CAAMR group compared to three other histology groups (normal, interstitial fibrosis and tubular atrophy, and calcineurin inhibitors toxicity) after KTx. On the other hand, current clinical laboratory data could not differentiate the CAAMR group from these groups. These data suggest that urinary exosomal SYT17 is a potential diagnostic marker for CAAMR.

We prospectively evaluated refractive changes in the eyes of 97 patients who underwent trabeculectomy at Fukui University Hospital, Fukui, Japan. The primary outcome measure was the refractive change after trabeculectomy. Secondary outcome measures included postoperative complications and prognostic factors for refractive change. We observed a progressive and significant mean refractive myopic shift of −0.80 D at 12 months after surgery. In phakic eyes, the mean myopic refractive shifts progressed significantly by −0.46 D at 3 months after surgery (P=0.003), by −0.52 D at 6 months (P=0.012), and by −1.31 D at 12 months (P<0.001). In the pseudophakic eyes, we found no significant refraction progression at any of the postsurgery follow-up visits. Our multivariable analyses showed that lens nuclear color grade change was a significant prognostic factor for refractive myopic progression (P<0.001). Trabeculectomy causes refractive myopic progression in phakic eyes. Nuclear sclerotic cataract progression is associated with refractive myopic shift after trabeculectomy. This trail is registered with UMIN000007813.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by communication disability with no curative treatment. Maternal separation-induced ultrasonic vocalisation (USV) was widely used to assess communication disability between pups and dams. Particularly, USV calls in many genetically modified ASD model mice were altered. Previously, we demonstrated that mice pups exposed to valproic acid in utero (VPA pups) showed decreased number of USV calls on postnatal day 11 and were rescued by subcutaneous injection of oxytocin. However, the qualitative change of USV calls by oxytocin has not been evaluated in VPA pups. In the present study, we examined the duration of oxytocin effect and analysed the altered pattern of USV calls using VPA pups. The oxytocin administration increased the total number of USV calls and the effect persisted up to 120 min in VPA pups. The pattern analysis revealed that the increase in the number of complex calls also persisted up to 120 min. These results suggested that oxytocin had a prolonged effect on USV calls, mainly on complex calls, in VPA pup, showing that oxytocin could recover their social modality to respond to maternal separation.

The brain metastasis of lung cancer is a crucial problem that causes poor clinical outcome and alters patient's quality of life. Previous evidences showed that cancer cells may escape from host immunity to form distant metastasis. However, the detailed interaction between lung cancer cells and immune cells in the brain niche is still unclear. Microglia and perivascular macrophages are the only resident immune cells in the brain parenchyma in physiological state. Here, we develop an in-vivo imaging method that can allow us to follow the immune cells and cancer progression in single cell resolution to evaluate immune reaction against cancer. Using this method, we evaluated microglial role in the development of brain-metastasis.Lung cancer cell line (CMT167mC) expressing mCherry was established from CMT167, a mice lung cancer cell line derived from C57BL/6. To generate brain-metastasis model mice, the cancer cells were injected via the internal carotid artery of the CX3XR1-GFP mice (Day 0), in which EGFP is specifically expressed in microglia and macrophage. The skull of the mice was replaced with a glass coverslip for chronic imaging (Day 1), and the fluorescently labeled cancer cells and microglia was simultaneously visualized using two-photon microscopy (Day 2-). The median survival of the brain-metastasis model mice after cancer cell injection was 15.0 days, and the fate of each embolized cell was followed for 14 days. Monitoring of 277 embolized cancer cells in the brain revealed 49.8% of cancer cells displaced from their original site, 40.4% developed micro-metastasis (MMs), and 6.9% were phagocytosed by microglia. We focused on the microglial role around the cancer cells that can form MMs, then the morphology and behavior of microglia around MMs was evaluated. The size of microglia around MMs (100 µm) cancer cells, suggesting that cancer cells can escape from activated microglia without being phagocytosed. Microglia depletion using pexidartinib can reduce the number and size of MMs that implicates activated microglia can inversely help tumor progression. Then we evaluated the functional role of a “Don't eat me” signal against microglia, which is a surface protein CD47 that inhibit the phagocytic action of macrophages. A knockout of the CD47 gene from CMT167mC significantly reduced the formation of MMs, increased phagocytosis by microglia, and prolonged the survival time of the model mice. The result suggests that this model provide a method to visualize immune reaction against cancer and validation of crucial target that reduce MMs. We are further exploring another critical factor that regulate development of brain metastasis using multi-omics analysis both on the activated microglia and on the cancer cells that formed MMs. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Real time in vivo imaging of brain metastasis visualized dynamic reaction of microglia against cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1768.

ROS1 gene arrangements occur in approximately 1% to 2% of non-small cell lung cancers. Crizotinib, the ROS1-tyrosine kinase inhibitor (TKI), has been approved for ROS1-rearranged lung cancer based on its dramatic therapeutic effect, but cures are usually not achieved. We have been focused on relationships between Yes associated protein1 (YAP1) and resistance to molecular target drugs. YAP1, a main mediator of the Hippo pathway, promotes cell proliferation and epithelial-mesenchymal transition and is associated with drug resistance in some cancer types. We previously demonstrated that YAP1 mediates survival against alectinib therapy in anaplastic lymphoma kinase-rearranged lung cancer cells. Then, in this study, we evaluated the role of YAP1 as an initial survival mechanism from lorlatinib in ROS1-rearranged lung cancer. We established a patient-derived EZR-ROS1-rearranged lung cancer cell line (KTOR71) from pleural effusion of a patient who acquired resistance to crizotinib. Subsequently, we conducted functional analysis of YAP1 involvement in drug-resistance, using this patient-derived cell line. DNA sequencing confirmed that KTOR71 cells harbored EZR-ROS1 fusion and ROS1 S1986F mutation, which is known crizotinib resistance mutation. KTOR71 cells were sensitive to lorlatinib, while resistant to crizotinib in cell viability assay (CVA). Nuclear localization of YAP1, which is an activation marker of YAP1, was assessed using immunofluorescence staining. In KTOR71 cells, YAP1 was activated after lorlatinib exposure. Then, to evaluate functional role of YAP1 in the initial survival against lorlatinib, the genetic inhibition of YAP1 in KTOR71 cells were performed using small interfering RNA (siRNA). Silencing of YAP1 by siRNA enhanced sensitivity to lorlatinib in KTOR71 cells in CVA. Furthermore, to confirm this functional role of YAP1 in vivo, we established the KTOR71 cell-derived xenograft model. The combination therapy with lorlatinib and verteporfin, an inhibitor of YAP1-TEAD interaction, sustained tumor remission significantly compared with lorlatinib monotherapy in vivo. Finally, we found that AKT was reactivated with activation of YAP1 and silencing of YAP1 by siRNA suppressed this AKT reactivation. These results suggest that YAP1 may be a potential drug target for ROS1-rearranged lung cancer. Citation Format: Masatoshi Yamazoe, Hiroaki Ozasa, Tetsuya Ohgimoto, Kazutaka Hosoya, Hitomi Ajimizu, Tomoko Funazo, Yuto Yasuda, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Young Hak Kim, Hirai Toyohiro. Activation of YAP1 confers ROS1 inhibitor resistance in ROS1-rearranged lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1098.

Tumor formation is promoted by increased expression of “Don’t eat me” signals such as CD47 in cancer cells that inhibit phagocytic action of macrophages. However, the signals can regulate generation of brain metastasis is still unclear. In the brain, microglia and perivascular macrophages are the only resident immune cells of the brain parenchyma. Here, we have developed an in-vivo imaging method that can allow us to evaluate microglial phagocytosis of tumor and to detemine the fates of cancer cells in the brain. The skull of the CX3CR1-EGFP mice, in which microglia is specifically labeled with EGFP, was replaced with a glass coverslip, and the fluorescently labeled lung cancer cells (CMT167-mCherry) were injected via the internal carotid artery of the mice. The fate of arrested cancer cells in the brain vascular was monitored for 12-16 days. Of the 324 cancer cells (4 mice), 49.8% (N=138) of cells displaced from their original site, 40.4% (N=112) of cells developed micro-metastasis, and 6.9% (N=27) of cells were processed by microglia resulting in cell death. To evaluate the “Don’t eat me” signal against microglia, a CD47 gene knockout cell line was monitored as well, and these cells developed significantly less micro-metastasizes compared with CD47 positive cells. This in vivo imaging of the brain metastasis potentially provide a method for monitoring of tumor fate and validation of crucial target. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Novel in vivo imaging method to evaluate "Don't eat me" signal of tumor against microglia [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO039.

The ribosomal protein L11 (RPL11) binds and inhibits the MDM2 ubiquitin ligase, thereby promoting p53 stability. Thus, RPL11 acts as a tumor suppressor. Here, we show that GRWD1 (glutamate‐rich WD40 repeat containing 1) physically and functionally interacts with RPL11. GRWD1 is localized to nucleoli and is released into the nucleoplasm upon nucleolar stress. Silencing of GRWD1 increases p53 induction by nucleolar stress, whereas overexpression of GRWD1 reduces p53 induction. Furthermore, GRWD1 overexpression competitively inhibits the RPL11–MDM2 interaction and alleviates RPL11‐mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N‐terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage‐independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene. ![][1] The ribosomal protein L11 (RPL11) functions as a tumor suppressor by inhibiting the MDM2 ubiquitin ligase that promotes degradation of p53. This study shows that GRWD1 (glutamate‐rich WD40 repeat containing 1) binds to RPL11, thereby counteracting RPL11‐mediated suppression of MDM2, and down‐regulating p53 levels. [1]: /embed/graphic-1.gif