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An 11-month-old boy with productive cough was referred to our hospital. He had nasal obstruction immediately after birth, and wheezing, wet cough, and rhinorrhea were observed daily after the neonatal period. Clinical and imaging findings revealed secretory otitis media, chronic sinusitis, and bronchiectasis. Primary ciliary dyskinesia was suspected. Transmission electron microscopy of nasal cilia showed defects of the outer and inner dynein arms. Genetic examinations of the family revealed copy number variation in PIH1 domain-containing 3 (PIH1D3) in the proband and mother. This is the first report of a Japanese patient with primary ciliary dyskinesia caused by copy number variation in PIH1D3.

Change-N1 peaking 90-180 ms after changes in a sound feature of a continuous sound is clearly attenuated by a preceding change stimulus (called a "prepulse"). Here, we investigated the effects of a preceding decrease in sound pressure on the degree of inhibition of the subsequent Change-N1 amplitude. Using 100-Hz click train sounds, we obtained Change-N1s from 11 healthy volunteers. The two types of test stimuli were an abrupt 10-dB increase from the baseline (70 dB) and the insertion of a 0.45-ms inter-aural time difference in the middle of the sound. Three consecutive clicks at 30, 40, and 50 ms before the change onset that was used as a prepulse were weaker than the background by 5 or 10 dB. The Change-N1 elicited by the two test stimuli was attenuated more strongly by the weaker prepulse, which was not congruent with the theory that the inhibition of the subsequent sensory/sensory-motor processing depends on the sound pressure level of a prepulse. These results suggest that a change in any type of sound feature elicits a change-related response that is inhibited by any type of preceding change stimulus, which reflects auto-inhibition of the change-responding circuit.

Multifaceted analysis is recommended for the diagnosis of primary ciliary dyskinesia (PCD). A 31-year-old woman had situs inversus, bronchiectasis, family history of PCD, and compound heterozygous mutations in DNAH5. Her cilia were immotile. Defects in the outer dynein arms were revealed by transmission electron microscopy and loss of DNAH5 proteins in the entire length of axonemes using immunofluorescence (IF). A 17-year-old boy had bronchiectasis and heterozygous mutations in DNAH11. His cilia were motile with normal ultrastructure. The loss of DNAH11 proteins at the proximal region of cilia was revealed by IF. IF could be useful to support PCD diagnosis.

Myosteatosis is gathering attention as a feasible indicator for sarcopenia and increased risk of morbidity. However, the prognostic value of intramuscular adipose tissue content (IMAC) as an assessment method for myosteatosis remains controversial. The objectives of this study are to compare the prognostic value of intramuscular adipose tissue content (IMAC) with our newly-developed modified IMAC (mIMAC), and to assess the clinical significance of mIMAC in colorectal cancer (CRC) and gastric cancer (GC).We evaluated 892 patients with CRC or GC, and assessed preoperative IMAC and mIMAC to compare their prognostic and predictive values for postoperative infectious complications in both cohorts.Both preoperative IMAC and mIMAC were sex- and disease-dependent, and positively or negatively correlated with age in CRC and GC patients (IMAC: CRC: r = 0.33, P 0.0001; GC: r = 0.304, P 0.0001; mIMAC: CRC: r = -0.364, P 0.0001; GC: r = -0.263, P 0.0001). In contrast to IMAC, lower preoperative mIMAC was significantly associated with disease-development factors, and was an independent prognostic factor for both overall survival (OS) and disease-free survival (DFS) in both CRC (OS: hazard ratio (HR): 1.95, 95% confidence interval (CI): 1.25-3.03, p = 0.003; DFS: HR: 1.93, 95% CI: 1.22-3.04, p = 0.005) and GC patients (OS: HR: 2.11, 95% CI: 1.22-3.68, P = 0.008; DFS: HR: 2.03, 95% CI: 1.18-3.5, P = 0.011). Patients with postoperative remote infections had a poorer prognosis compared with those without in both cohorts (CRC: HR: 2.67, 95% CI: 1.46-4.89, P = 0.002; GC: HR: 3.01, 95% CI: 1.47-6.19, P = 0.003), and low mIMAC was an independent risk factor for postoperative remote infection in both cancers (CRC: odds ratio (OR): 2.56, 95% CI: 1.06-6.23, P = 0.038; GC: OR: 2.8, 95% CI: 1.03-7.58, P = 0.043). Finally, we assessed the correlation between IMAC or mIMAC and the representative frailty markers body mass index (BMI), serum albumin, and prognostic nutritional index (PNI). We found a positive correlation between preoperative mIMAC and all of these markers in both cohorts (CRC: BMI: r = 0.193, P 0.0001; serum albumin: r = 0.42, P 0.0001; PNI: r = 0.39, P 0.0001; GC: BMI: r = 0.22, P 0.0001; serum albumin: r = 0.212, P 0.0001; PNI: r = 0.287, P 0.0001).Preoperative mIMAC could be useful for perioperative and postoperative management in CRC and GC.

MPL exon 10 mutations are one of the driver mutations in essential thrombocythemia (ET) or myelofibrosis (MF). We have established an in-house MPL mutation analysis system, covering the entire region of MPL exon 10 by direct sequencing. Since 2009, MPL exon 10 mutation analysis has been performed for diagnosis of myeloproliferative neoplasms (MPN) without JAK2 V617F or CALR exon 9 mutations. So far, 11 cases of MPL exon 10 mutation have been found in 51 patients with suspected MPN. In patients with ET, we detected five non-canonical MPL mutations including one novel mutation, MPL R514_P518delinsK, and one canonical MPL W515L mutation. Notably, three ET patients without canonical MPL mutations had thrombotic events. Meanwhile, in primary or secondary MF, only canonical MPL W515L/K mutations were found. Further cases need to be examined to elucidate the full MPL mutation profile in MPN. However, our data indicate that analysis of the whole of MPL exon 10 is warranted for the diagnosis of MPL mutations, especially in ET, and that the use of Japanese commercial laboratory tests that only detect canonical MPL W515L/K mutations may miss a significant percentage of MPL exon 10 mutations, which could delay the administration of anti-thrombotic therapy.

Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first‐line chemotherapy to assess the benefit of anti–epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)‐based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti–EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next‐generation sequencing‐based tests are weakly recommended because these tests have not been validated in clinical trials., Update to the molecular testing guideline for colorectal cancer defined by Japanese Society of Medical Oncology. The appropriate timing of each test and degree of recommendation are summarized.

The management of secondary findings (SFs), which are beyond the intended purpose of the analysis, from clinical comprehensive genomic analysis using next generation sequencing (NGS) presents challenges. Policy statements regarding their clinical management have been announced in Japan and other countries. In Japan, however, the current status of and attitudes of clinical genetics professionals toward reporting them are unclear. We conducted a questionnaire survey of clinical genetics professionals at two time points (2013 and 2019) to determine the enforcement of the SF management policy in cases of comprehensive genetic analysis of intractable diseases and clinical cancer genome profiling testing. According to the survey findings, 40% and 70% of the respondents stated in the 2013 and 2019 surveys, respectively, that they had an SF policy in the field of intractable diseases, indicating that SF policy awareness in Japan has changed significantly in recent years. Furthermore, a total of 80% of respondents stated that their facility had established a policy for clinical cancer genome profiling testing in the 2019 survey. In both surveys, the policies included the selection criteria for genes to be disclosed and the procedure to return SFs, followed by recommendations and proposals regarding SFs in Japan and other countries. To create a better list of the genes to be disclosed, further examination is needed considering the characteristics of each analysis.

To the best of our knowledge, there are no useful screening methods for early detection of endometrial cancer in asymptomatic individuals. The present study evaluated the usefulness of genetic analysis of liquid-based cytology (LBC) specimens by assessing whether pathological genetic mutations detected in cancer tissue sections were detected in LBC specimens from the cervix and uterus. The primary endpoint was genetic analysis of cervical cytology specimens and LBC for the detection of endometrial cancer. Endometrial thickening (11 mm) assessed using transvaginal ultrasonography was present in 60% of cases and adenocarcinoma assessed using cervical cytology was present in 50% of cases. In 70% of cases, pathogenic mutations detected in cancer tissue sections were also detected in cervical and/or endometrial LBC specimens. The pathogenic variants identified were

Primary ciliary dyskinesia (PCD) is diagnosed through multiple methods, including transmission electron microscopy (TEM), a high-speed video microscopy analysis (HSVA), immunofluorescence (IF), and genetic testing. A primary cell culture has been recommended to avoid the misdiagnosis of secondary ciliary dyskinesia derived from infection or inflammation and improve diagnostic accuracy. However, primary cells fail to differentiate into ciliated cells through repeated passages. The conditional reprogramming culture (CRC) method, a combination of a Rho-kinase inhibitor and fibroblast feeder cells, has been applied to cystic fibrosis. The goal of this study was to evaluate the value of CRC in diagnosing PCD in Japanese patients.Eleven patients clinically suspected of having PCD were included. Airway epithelial cells were obtained from an endobronchial forceps biopsy and cultured at the air-liquid interface (ALI) combined with CRC. Ciliary movement, ultrastructure, and mutated ciliary protein evaluation were performed using HSVA, TEM, and IF, respectively. Genetic testing was performed on some patients.CRC yielded dense and well-differentiated ciliated cells with a high success rate (∼90%). In patients with PCD, the ciliary ultrastructure phenotype (outer dynein arm defects or normal ultrastructure) and IF findings (absence of the mutated ciliary protein) were confirmed after CRC. In DNAH11-mutant cases with normal ultrastructure by TEM, the HSVA revealed stiff and hyperfrequent ciliary beating with low bending capacity in CRC-expanded cells, thereby supporting the diagnosis.CRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared 5 samples from patients with lung or colorectal cancer, extracted genomic DNA from the cancer tissue and peripheral blood, and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants, and 25-bp delins in the EGFR exon 19, not identified by the in vitro diagnostics testing. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. All NGS platforms and bioinformatics pipelines have advantages and disadvantages. We propose the use of a PT program using patient samples to ascertain the quality status of cancer gene testing in laboratories and to ensure that laboratories have sufficient information to develop advancements in precision medicine for cancer.

We report the first Japanese case of primary ciliary dyskinesia caused by DNAH9 variations. The patient, a 5-year-old girl, had repeated episodes of productive cough after contracting the common cold at the age of 1 year and 6 months. She did not have a situs abnormality or congenital heart defect. We identified two novel DNAH9 variants, NM_001372.3: c. [1298C>G];[5547_5550delTGAC], (p.[Ser433Cys];[Asp1850fs]).

Objective Primary ciliary dyskinesia (PCD) is a rare hereditary disease. Most reports of PCD in Japan are case reports, and clinical analysis has not been performed. Differences in the causative genes might affect the clinical features in different ethnic groups. The purpose of this study was to clarify the clinical features of Japanese patients with PCD. Methods We performed a retrospective chart review of PCD patients seen at Mie University Hospital and patients whose blood samples were sent to us for genetic analysis from 2011 to 2020. Data on the following items were collected and analyzed: age at first visit to the hospital, age at diagnosis of PCD, process of referral to our facility, chief complaint, situs status, PrImary CiliARy DyskinesiA Rule (PICADAR) score, nasal nitric oxide concentration, otoscopic findings, rhinoscopic findings, and paranasal computed tomography scan findings. Results Sixty-seven patients (24 male, 43 female) were diagnosed with PCD during the study period. Age at diagnosis ranged from 2 months to 69 years (median, 17 years). Respiratory symptoms (77%) were the most common complaint, followed by nasal (15%) and aural (8%) symptoms. Situs inversus was present in 17 (25%) cases. Only 2 cases had congenital cardiac anomalies. The mean PICADAR score was 7.3 (range, 3-14) points. Approximately 50% of tympanic membranes showed retraction, suggesting otitis media with effusion. The mean Lund-Mackay score was 12.8 (range, 7-17) points, suggesting that the radiographic findings were not always severe. There was no significant difference in the total Lund-Mackay score between patients with and without situs inversus (12.7 vs. 12.6, respectively). Conclusion Situs inversus was present in 25% of Japanese PCD patients, which is much lower than observed in other countries. This is a result of differences in the major disease-causing genes. The general rule that "situs inversus is observed in approximately 50% of PCD patients" cannot be applied, at least, in Japanese PCD patients.

Microsatellite instability-high (MSI-H) is an important biomarker for predicting the effect of immune-checkpoint inhibitors (ICIs) on advanced solid tumors. MSI-H is detected in various cancers, but its frequency varies by cancer type and stage. Therefore, precise frequency is required to plan ICIs therapy. In this study, the results of MSI test actually conducted in clinical practice were investigated. In total, 26,469 samples of various cancers were examined to determine if PD-1 blockade was indicated between December 2018 to November 2019. The results of MSI test were obtained for 26,237 (99.1%) among them. The male to female ratio was 51:49 and mean age was 64.3 years. In all the samples, overall frequency of MSI-H was 3.72%. By gender, the frequency of MSI-H was higher in female patients (4.75%) than in male patients (2.62%) (P0.001). A comparison by age revealed that the frequency of MSI-H was significantly higher in patients aged less than 40 years (6.12%) and 80 years or older (5.77%) than in patients aged between 60 and 79 years (3.09%) (P0.001). MSI-H was detected in 30 cancer types. Common cancer types were endometrial cancer, 16.85%; small intestinal cancer, 8.63%; gastric cancer, 6.74%; duodenal cancer, 5.60%; and colorectal cancer, 3.78%. MSI-H was detected in cancer derived from a wide variety of organs. The frequency of MSI-H varied by cancer types and onset age. These data should prove especially useful when considering ICI treatment. Supporting Information.

Systemic inflammatory response influences cancer development and perioperative surgical stress can affect the survival of patients with colorectal cancer (CRC). We developed a system to cumulatively assess perioperative inflammatory response and compare the prognostic value of various cumulative inflammatory and nutritional markers in patients with CRC. We assessed perioperative cumulative markers using the trapezoidal area method in 307 patients who underwent surgery for CRC and analyzed the results statistically. The cumulative lymphocyte to C-reactive protein (CRP) ratio (LCR) predicted survival more accurately than other well-established markers (sensitivity: 80.0%, specificity: 69.3%; area under the curve (AUC): 0.779; P

MPL exon 10 mutations are one of the driver mutations in essential thrombocythemia (ET) or myelofibrosis (MF). We have established an in-house MPL mutation analysis system, covering the entire region of MPL exon 10 by direct sequencing. Since 2009, MPL exon 10 mutation analysis has been performed for diagnosis of myeloproliferative neoplasms (MPN) without JAK2 V617F or CALR exon 9 mutations. So far, 11 cases of MPL exon 10 mutation have been found in 51 patients with suspected MPN. In patients with ET, we detected five non-canonical MPL mutations including one novel mutation, MPL R514_P518delinsK, and one canonical MPL W515L mutation. Notably, three ET patients without canonical MPL mutations had thrombotic events. Meanwhile, in primary or secondary MF, only canonical MPL W515L/K mutations were found. Further cases need to be examined to elucidate the full MPL mutation profile in MPN. However, our data indicate that analysis of the whole of MPL exon 10 is warranted for the diagnosis of MPL mutations, especially in ET, and that the use of Japanese commercial laboratory tests that only detect canonical MPL W515L/K mutations may miss a significant percentage of MPL exon 10 mutations, which could delay the administration of anti-thrombotic therapy.

Microsatellite instability‐high (MSI‐H) is an important biomarker for predicting the effect of immune checkpoint inhibitors (ICIs) on advanced solid tumors. Microsatellite instability‐high is detected in various cancers, but its frequency varies by cancer type and stage. Therefore, precise frequency is required to plan ICI therapy. In this study, the results of MSI tests actually carried out in clinical practice were investigated. In total, 26 469 samples of various cancers were examined between December 2018 and November 2019 to determine whether programmed cell death‐1 blockade was indicated. The results of MSI tests were obtained for 26 237 (99.1%) of these samples. The male : female ratio was 51:49 and mean age was 64.3 years. In all samples, the overall frequency of MSI‐H was 3.72%. By gender, the frequency of MSI‐H was higher in female patients (4.75%) than in male patients (2.62%; P, In this study, we presented real‐world data on microsatellite instability status in various unresectable or metastatic solid tumors to determine if pembrolizumab treatment was indicated.

Background We compared achievement rate of sufficient tacrolimus blood concentration in the early postoperative period and incidence of acute cellular rejection within 1 month after living donor liver transplantation (LDLT) between tacrolimus intravenous (IV) and oral administration groups. Methods From October 2005 to November 2016, 61 LDLT patients administered tacrolimus, who could be genotyped for CYP3A5*3 and *1, were chosen from the electronic record database. The patients were then divided into the 2 groups (an IV group [n = 38] and an oral group [n = 23]). We defined patients with 1*1 or *1*3 as expressors and those with *3*3 as nonexpressors. Sufficient trough level tacrolimus blood concentration on postoperative day (POD) 3 was defined as 10–20 ng/mL. Results Comparable concentrations were seen between the 2 groups, with mean blood concentration 13.7 ± 8.5 ng/mL in the oral group and 15.2 ± 4.3 ng/mL in the IV group. Achievement rate of sufficient tacrolimus concentration on POD 3 was significantly higher in the IV group than in oral group: 97% (37 of 38) vs 65% (15 of 23), respectively ( P = .001). When we focused on achievement rate in the oral group according to CYP3A5 polymorphism, the frequency of expressors (17%) was significantly lower than that of nonexpressors (82%) ( P = .016). However, in the IV group this negative influence was totally eliminated, resulting in high achievement rates regardless of CYP3A5 polymorphism. In terms of incidence of acute cellular rejection, there was no significant difference between the 2 groups (IV 32% vs oral 17%, P = .250). Conclusion IV administration of tacrolimus allowed us to obtain more stable control of blood concentration regardless of CYP3A5 genotype.

We explored the relationship between abdominal vein thromboses, including portal vein thrombosis (PVT) and superior mesenteric vein thrombosis (SMVT), and thrombophilia. The frequency of thrombophilia, such as antithrombin (AT), protein C (PC), or protein S (PS) gene mutations, was examined in 21 patients with PVT, 6 patients with SMVT, and 6 patients with both PVT and SMVT. Low levels of AT, PC, or PS were frequently detected in patients with PVT or mesenteric vein thrombosis, and 4 mutations in the PS gene, 3 mutations in the PC gene, and 2 mutations in AT the gene were detected. Protein S Tokushima was detected in 3 of 4 patients with a PS gene mutation and was associated with 2 other PS gene mutations. The onset of PVT or SMVT was almost idiopathic in patients with congenital thrombophilia. Both PVT and SMVT were frequently caused by an AT, a PC, or a PS mutation, and the onset of these thromboses due to thrombophilia was frequently idiopathic.

Background Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The involvement of copy number variation (CNV) in the development of PCD is largely unknown. Methods We examined 93 Japanese patients with clinically suspected PCD from 84 unrelated families. CNV was examined either by exome sequencing of a PCD gene panel or by whole‐exome sequencing (WES). The identified alterations were validated by PCR and Sanger sequencing. Nasal ciliary ultrastructure was examined by electron microscopy. Results Analysis of CNV by the panel or WES revealed a biallelic deletion in the dynein regulatory complex subunit 1 (DRC1) gene in 21 patients, which accounted for 49% of the PCD patients in whom a disease‐causing gene was found. Sanger sequencing of the PCR product revealed a 27,748‐bp biallelic deletion including exons 1–4 of DRC1 with identical breakpoints in all 21 patients. The ciliary ultrastructure of the patients with this CNV showed axonemal disorganization and the loss or gain of central microtubules. Conclusion The deletion of DRC1 is the major cause of PCD in Japan and this alteration can cause various ciliary ultrastructural abnormalities., Analysis of copy number variation revealed a biallelic deletion in the dynein regulatory complex subunit 1 (DRC1) gene in 21 patients, which accounted for 49% of the primary ciliary dyskinesia patients in whom a disease‐causing gene was found. Sanger sequencing of the PCR product revealed a 27,748‐bp biallelic deletion including exons 1–4 of DRC1 with identical breakpoints in all 21 patients. The ciliary ultrastructure of the patients with this copy number variation showed axonemal disorganization and the loss or gain of central microtubules.

The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorders and schizophrenia. Various screening methods have been used to assess the developmental neurotoxicity (DNT) of chemicals, with most assays focusing on cell viability, apoptosis, proliferation, migration, neuronal differentiation, and neuronal network formation. However, assessment of toxicity during progenitor cell differentiation into neurons, astrocytes, and oligodendrocytes often requires immunohistochemistry, which is a reliable but labor-intensive and time-consuming assay. Here, we report the development of a triple-transgenic zebrafish line that expresses distinct fluorescent proteins in neurons (Cerulean), astrocytes (mCherry), and oligodendrocytes (mCitrine), which can be used to detect DNT during neuronal differentiation. Using in vivo fluorescence microscopy, we could detect DNT by 6 of the 10 neurotoxicants tested after exposure to zebrafish from 12 h to 5 days&rsquo, post-fertilization. Moreover, the chemicals could be clustered into three main DNT groups based on the fluorescence pattern: (i) inhibition of neuron and oligodendrocyte differentiation and stimulation of astrocyte differentiation, (ii) inhibition of neuron and oligodendrocyte differentiation, and (iii) inhibition of neuron and astrocyte differentiation, which suggests that reporter expression reflects the toxicodynamics of the chemicals. Thus, the triple-transgenic zebrafish line developed here may be a useful tool to assess DNT during neuronal differentiation.

To determine whether an intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) in eyes with diabetic macular edema (DME) affects the vascular infarction-related molecules (VIRMs). Nineteen eyes with DME were treated with 0.5 mg of intravitreal ranibizumab (IVR), and 22 eyes with DME were treated with 2 mg of intravitreal aflibercept (IVA). Blood was collected before, 1 week and 1 month after the injections. Aqueous humor was collected before and 1 month after the injections. The concentration of the VIRMs (cardiac myoglobin, cardiac troponin, intercellular adhesion molecule, monocyte chemotactic protein-1, matrix metalloproteinase-8, placental growth factor [PlGF], tenascin-C, tissue inhibitor of metalloproteinase-1, thrombospondin-2, vascular cell adhesion molecule-1, and VEGF) were determined by the multiplex assay. After the single injection of both types of anti-VEGF agents, the concentration of aqueous VEGF decreased significantly (P P

Background: Gastric cancer (GC) is one of the most common cancers and leading cause of cancer-related deaths worldwide, primarily because of rapid disease progression to advanced stages and highly malignant potential. Methyltransferase-like 3 (METTL3) is a crucial component of the m6A methyltransferase complex, and plays pivotal roles in tumour progression. This study investigated the prognostic significance of METTL3 expression in gastric cancer (GC). Methods: We assessed expression levels of METTL3 by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) tissue specimens from one hundred fifty-eight GC patients. Propensity score matching (PSM) analysis was performed to clarify its prognostic potential. The METTL3 gene expression was also investigated in fresh frozen specimens from another independent cohort of fifty-seven GC patients to establish its clinical relevance. Knockdown of METTL3 by siRNA transfection was performed to evaluate its function in vitro. Results: METTL3 expression was significantly higher in cancerous tissues vs. corresponding normal mucosa (P Conclusion: METTL3 expression may be used as a clinically feasible prognostic marker and could serve as a potential therapeutic target in GC patients. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Chengzeng Yin, Ma Ruiya, Akul Goel, Takashi Ichikawa, Hiroki Imaoka, Takahito Kitajima, Tadanobu Shimura, Mikio Kawamura, Hiromi Yasuda, Hiroyuki Fujikawa, Takeshi Yokoe, Ikuyo Mochiki, Masaki Ohi, Kaname Nakatani. Clinical value and oncogenic role of METTL3 expression in gastric cancer patients [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 653.

An abrupt change in a sound feature (Test) in a continuous sound elicits an auditory evoked potential, peaking at approx. 100–180 ms (Change-N1) after the change onset. Change-N1 is attenuated by a preceding weak change stimulus (Prepulse), in the phenomenon known as prepulse inhibition (PPI). In this electroencephalographic study, we compared these two indexes among scalp electrodes. Change-N1 was elicited by an abrupt 10-dB increase in sound pressure in repeats of a 70-dB click sound at 100 Hz and was recorded using 22 electrodes in 31 healthy subjects. The prepulse was a 10-dB decrease in three consecutive clicks at 30, 40, and 50 ms before the Test onset. Four stimuli (Test alone, Test with Prepulse, Prepulse alone, and background alone) were presented randomly through headphones at an even probability. The results demonstrated that: (1) Electrodes at the frontal/central midline were reconfirmed to be suitable to record Change-N1; (2) Change-N1 showed right-hemisphere predominance; (3) There was no difference in the %PPI among regions (prefrontal/frontal/central) and hemispheres (midline/left/right); and (4) the Change-N1 amplitude and its PPI at prefrontal electrodes were positively correlated with those at the frontal electrodes. These results support the use of Change-N1 and its PPI as a tool to evaluate the change detection sensitivity and inhibitory function in individuals. The use of prefrontal electrodes can be an option for a screening test.

Although prognosis in patients with localized osteosarcoma has been dramatically improved by the introduction of multiple chemotherapy agents known as combination chemotherapy, there is growing concern about the development of secondary malignant neoplasms. We report the case of a 13-year-old girl in whom the diagnosis of Ewing sarcoma of bone localized on the shaft of left femur was made 2 years after successful treatment without radiotherapy for osteosarcoma of right proximal femur. EWS-FLI1 fusion gene was detected by reverse transcriptase-polymerase chain reaction. To our knowledge, this is the first case with Ewing sarcoma of the bone as a secondary malignant neoplasm developed in osteosarcoma survivor. We collected 15 cases, included this case, with secondary Ewing sarcoma family of tumor by utilizing the PubMed search and might consider the causes of this secondary cancer.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by structural and/or functional impairment of cilia throughout the whole body. Early diagnosis of PCD is important for the prevention of long‑term sequelae, however early diagnosis is a challenge due to the phenotypic heterogeneity of PCD. In the current study, the patient with PCD was diagnosed at nine years old following several efforts to control intractable airway symptoms. The patient experienced a chronic productive cough beginning in early childhood and had multiple episodes of pneumonia and otitis media with effusion and sinusitis. No situs inversus or other heterotaxias were reported. Serial chest X‑rays exhibited persistent atelectasis and bronchiectasis in the right middle lobe. When the patient was nine years old, electron microscopy of his cilia and genetic analysis were conducted. Electron microscopy of a biopsy specimen from the nasal mucosa indicated loss of the outer dynein arms. Whole‑exome analysis of the genome demonstrated the presence of compound heterozygous mutations in DNAH5: NM_001369.2:c.5983C>T, p.Arg1995X in exon 36 and NM_001369.2:c.9101delG, p.Gly3034ValfsX22 in exon 54; neither of which have been previously reported in the literature in a Japanese patient. Notably, this case is, to the best of our knowledge, the first reported case of PCD caused by the DNAH5 mutation in a Japanese patient.

We analyzed a cohort of 36 females with pregnancy loss. In addition to 11 patients with antiphospholipid antibody syndrome and 2 patients with congenital antithrombin (AT) or protein C deficiency, we identified 5 patients with low fibrinogen levels (median 110 mg/dL) prior to 10 weeks of gestation. Four of these 5 patients underwent a fibrinogen gene analysis, and all 4 were found to be heterozygotes for the α-fibrinogen (FGA) Thr321Ala polymorphism. One female without hypofibrinogenemia with a history of 8 pregnancy losses was found to be homozygous for the same polymorphism, and she also showed hypercoagulability without thrombosis. In conclusion, there was a relatively high frequency of pregnancy loss in the setting of hypofibrinogenemia and/or the FGA Thr312Ala polymorphism, and this may be an important risk factor for pregnancy loss and a hypercoagulable state in later pregnancy.

Primary ciliary dyskinesia (PCD) is a genetic disease inherited in an autosomal recessive manner. The prevalence of PCD is estimated to be 1 in 20,000 live births. Congenital abnormality of the primary cilia results in situs inversus in 50% of patients. Decreased function of motile cilia causes chronic rhinosinusitis, otitis media with effusion, bronchiectasis and infertility. Cases with situs inversus are considered to show "Kartagener's syndrome", and diagnosis is not difficult. However, in cases without situs inversus, the diagnosis is much more troublesome. PCD without situs inversus is thus probably underdiagnosed. Prolonged chronic cough represents an important symptom that is seen in most patients. The diagnosis of PCD requires the presence of the characteristic clinical phenotypes and either: (1) specific ciliary ultrastructural defects identified by transmission electron microscopy in biopsy samples of respiratory epithelium; or (2) identification of mutation in one of the genes known to be associated with PCD. Nasal nitric oxide concentration is extremely low in PCD, and this could be useful for screening of the disease. At present, no fundamental therapies are available for PCD. Diagnosis in the early stages is important to prevent progression of bronchiectasis and deterioration of lung function by guidance for daily life, immunization, cessation of smoking and prompt therapy at the time of respiratory tract infection. Since PCD is inherited in an autosomal-recessive manner, genetic counseling is necessary after definite diagnosis.

Several studies have reported risk factors for predicting cisplatin-induced acute kidney injury (AKI), including old age, female sex, smoking, hypoalbuminemia, hypokalemia, hypomagnesemia, a high body surface area, advanced cancer and the total dose of cisplatin administered. Recently, some studies have focused on the associations between genetic alterations in the genes coding for renal drug transporters, such as organic cation transporter 2 (OCT2), and the nephrotoxicity of cisplatin. However, genetic variants have not been fully elucidated for clinical use. Patients who had received cisplatin (≥50 mg/m2)-containing chemotherapy as a first-line treatment were considered as eligible for the present study. The occurrence of AKI and its associations with baseline characteristics, conventional biomarkers and single-nucleotide variants (SNV) were assessed. AKI was defined as an increase in the serum creatinine level of >0.3 mg/dl or to 1.5-2 times the baseline level. Genotyping was conducted using the DMET platform (DMET Plus), which characterizes 1,936 genetic variants (1,931 SNV and 5 copy number variations) in 231 genes. Between April 2014 and June 2016, a total of 28 patients (22 men and 6 women) were enrolled. AKI occurred in 8 of the 28 enrolled patients (28.6%). Univariate analyses demonstrated that the urinary β2-microglobulin level and body surface area were significantly higher in the AKI group (P

803 Background: The recent success of immunotherapy in high-frequency microsatellite instability (MSI-H) and/or mismatch repair protein deficient (MMR-D) tumors now supports testing for MSI in all advanced solid tumors. We previously reported using the quasi-monomorphic variation range (QMVR), the MSI status can be determined without matched normal DNA (Bando H, et al. Cancer Sci. 2018). Here, we investigate the proportion of the MSI status across various advanced solid tumors by this method. Methods: The MSI analysis was performed centrally by CLIA-certified, CAP-accredited, the single laboratory using tumor DNA from formalin-fixed paraffin embedded (FFPE) tissues with the MSI test kit (FALCO), which includes fluorescent-labelled primers for co-amplification of five mononucleotide markers (NR-21, BAT-25, MONO-27, NR-24 and BAT-26). The MSI-H was defined as positive if 2 or more makers showed instable. Results: A total of 18,250 patients with advanced solid tumors were registered from Dec 2018 to Aug 2019, and the MSI status was confirmed in 18,088 patients (99%). Among them, 51.9% were male, with median of 62.0 y/o. Overall prevalence of the MSI-H was3.3% (2.3% in male and 4.5% in female); the highest was 16.2% (149/916) in endometrial cancer, followed by 13% (13/100) in small intestinal cancer, 5.9% (74/1277) in gastric cancer, 3.3% (236/7190) in colorectal cancer, 2.6% (4/154) in prostate cancer, 2.5% (20/806) in ovarian cancer, 2.1% (9/426) in cervical cancer, 1.6% (13/831) in biliary tract cancer, 1.5% (4/271) in hepatocellular carcinoma, 0.9% (16/1852) in pancreas cancer. Interestingly, among biliary tract cancers, all MSI-H cases were arising from gall bladder (13/152) but no other sites. High prevalence was observed in cancer of teens or twenties (7.9%) and over 80y/o (5.6%), compare with others (3.0%) in this study (p < 0.001, both). Conclusions: These data show the MSI-H advanced solid tumors are distributed across a much broader tumor spectrum, and confirm the clinical relevance of the screening in patients with most advanced solid tumors, which can select patients sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.

The Japanese Society of Medical Oncology (JSMO) previously published 2 editions of the clinical guidelines: "Japanese guidelines for testing of KRAS gene mutation in colorectal cancer" in 2008 and "Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients" in 2014. These guidelines have contributed to the proper use of KRAS and RAS mutation testing, respectively. Recently, clinical utility, particularly for colorectal cancer (CRC) patients with BRAF V600E mutation or DNA mismatch-repair (MMR) deficiency, has been established. Therefore, the guideline members decided these genetic alterations should also be involved. The aim of this revision is to properly carry out testing for BRAF V600E mutation and MMR deficiency in addition to RAS mutation. The revised guidelines include the basic requirements for testing for these genetic alterations based on recent scientific evidence. Furthermore, because clinical utility of comprehensive genetic testing using next-generation sequencing and somatic gene testing of analyzing circulating tumor DNA has increasingly evolved with recent advancements in testing technology, we noted the current situation and prospects for these testing technologies and their clinical implementation in the revised guidelines.

BACKGROUND Blood tacrolimus (TAC) concentration delivered via intravenous administration is known to be influenced by genetic polymorphism of CYP3A5 and interaction with triazole antifungal agents. However, interindividual variability of blood TAC concentration is as of yet still difficult to predict during the early stages of hematopoietic stem cell transplantation (HSCT). This study was conducted to assess the wide variability of blood TAC concentrations because of the hepatic metabolic activities of CYP3A and CYP2C19 in HSCT recipients. METHODS This study is a single-institute prospective study that includes 21 adult patients who underwent HSCT and received 24 hours continuous intravenous administration of TAC at the Mie University Hospital between January 2009 and March 2014. After HSCT, the changes in blood TAC concentration/dose (C/D) ratio and TAC dose reduction from initial dose were investigated. RESULTS Significant differences between HSCT recipients with CYP3A5*1 allele and CYP3A5*3/*3 genotype were observed with respect to the median TAC C/D ratio on day 14 (563 versus 742 ng/mL per mg/kg, P < 0.01) and day 21 (672 versus 777 ng/mL per mg/kg, P < 0.05) after HSCT. Concomitant administration of voriconazole (VRCZ), but not of lansoprazole, was found to significantly increase the median TAC C/D ratio on day 14 (557 versus 723 ng/mL per mg/kg, P < 0.01). Possession of the CYP3A5*3/*3 genotype (day 14: odds ratio, 32.2; day 21: odds ratio, 33.0; P < 0.05) and concomitant administration of VRCZ (day 14: odds ratio, 37.8; P < 0.05) were found to be independent risk factors, which significantly contributed to an increased TAC C/D ratio. In HSCT recipients with CYP3A5*3/*3 genotype (78.0%), the median TAC dose ratio (day 21/day -1) was significantly lower compared with HSCT recipients with the CYP3A5*1 allele (94.1%), whereas VRCZ administration itself had no significant influence. Interestingly, in HSCT recipients with CYP2C19*1/*1, we found that the influence of VRCZ on the TAC dose ratio (85.7%) was relatively mild, even in a recipient with CYP3A5*3/*3. CONCLUSIONS In HSCT recipients, the variability of intravenous TAC concentration in the blood could be explained in part by the genetic variation of CYP3A5. The study results also strongly imply that the magnitude of hepatic interaction between TAC and VRCZ is affected by the genetic polymorphism of both CYP3A5 and CYP2C19 genes.

Objectives: To evaluate the influences of polymorphisms of cytochrome P450 (CYP) 3A5 (A6986G, CYP3A5*3) on serum levels of tacrolimus and cyclosporine (CyA) in patients with myasthenia gravis (MG). Methods: This study included 74 MG patients treated with tacrolimus (n=65) or CyA (n=22). Genomic DNA was extracted and amplified with specific primers, and CYP3A5 alleles were confirmed by direct sequencing of PCR products on an automated AB13100 DNA sequencer. We measured blood trough level (C0) of tacrolimus and CyA. Clinical disabilities were evaluated with the MG-ADL scale. Results: For tacrolimus C0, the CYP3A5*3/*3 genotype was associated with higher levels than the CYP3A5*1/*3 genotypes (7.1 ng/ml versus 2.9 ng/ml; P

To date, liquid biopsies have generated much interest as they involve minimally invasive blood tests, pro- vide an ongoing picture of a patient's cancer, and offer valuable insight into the best treatment. Liquid biop- sies detect circulating tumor cells (CTCs), fragments of tumor DNA and exosomes that are shed into the blood from the primary tumor and from metastatic sites. Liquid biopsies offer what tissue biopsies cannot due to risks to the patients and costs. Liquid biopsies allow the monitoring of genomic changes in tumors for the diagnosis of early and recurrent cancer and drug effects. In the future, instead of extensive imaging and invasive tissue biopsies, liquid biopsies could be used to guide cancer treatment decisions and even screen for tumors that are not vet visible on imaging.

Objective To evaluate otologic features of primary ciliary dyskinesia (PCD), especially eardrum features, audiometric findings, and clinical course. Study design Retrospective patient review. Setting Tertiary referral center. Patients Fifteen patients (mean age, 16.9 years [range, 1-32 yr]; 8 males and 7 females) diagnosed with PCD at our university hospital in the last 12 years. Intervention Diagnostic. Main outcome measures Electron microscopy of nasal cilia, gene mutation analysis, endoscopy of 30 eardrums, pure-tone audiometry, and tympanometry. Results All 15 patients showed ciliary ultrastructural abnormalities on electron microscopy and/or biallelic mutations in genes associated with ciliary function or structure. All 30 eardrums examined showed certain abnormalities. Fourteen patients had otitis media with effusion or its sequelae. The remaining patient had chronic otitis media. Pure-tone audiometry revealed the mean air conduction thresholds to be 25.0 and 26.4 dB in the right and left ears, respectively. In the ears with better hearing and worse hearing, the mean air conduction thresholds were 22.3 and 29.0 dB respectively. Conclusion Otologic disease among patients with PCD essentially comprised otitis media with effusion, and the patients' eardrums showed a variety of findings. Knowledge of these otologic features may lead to the early detection of PCD.

A total of 25 patients with autoimmune diseases receiving tacrolimus were screened using a peripheral blood cluster of differentiation 4+ adenosine triphosphate (ATP) activity assay (IMK assay) between October 2013 and July 2014. The autoimmune diseases of patients were as follows: Rheumatoid arthritis (n=15), lupus nephritis (n=6), ulcerative colitis (n=2) and myasthenia gravis (n=2). Patients were divided into two groups based on CYP3A5 genotype [expression of *1 allele: Expressor (EX; n=6) and non-expressor (NEX; n=19)]. The tacrolimus concentration and concentration/dose ratio was significantly lower in the EX group compared with the NEX group (P=0.0108 and 0.0056, respectively). In addition, all enrolled patients that presented with adverse effects belonged to the NEX group. No significant associations were observed between IMK ATP levels and the concentration or dose of tacrolimus (P=0.1092 and 0.6999, respectively). However, the IMK ATP high-level group exhibited a significantly higher occurrence rate of insufficient effect when compared with the normal and low-level groups (P=0.0014). In conclusion, the clearance of tacrolimus in patients with autoimmune diseases was affected by the CYP3A5 genotype, as previously reported in organ transplant patients. The IMK ATP level may indicate the clinical response irrespective of tacrolimus concentration.

The activated partial thromboplastin time (APTT) waveform includes several parameters that are related to various underlying diseases. The APTT waveform was examined in various diseases. Regarding the pattern of APTT waveform, a biphasic pattern of the first or second derivative curve (DC) was observed in patients with hemophilia and patients positive for antiphospholipid (aPL) antibodies or coagulation factor VIII (FVIII) inhibitors. The time of the first and second DC and fibrin formation at 1/2 height were prolonged in patients with hemophilia, patients with inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with disseminated intravascular coagulation (DIC). These values all tended to decrease in pregnant women (at 28-36 weeks’ gestation). The height of the second derivative peak 1 was significantly lower in patients with hemophilia, patients with FVIII inhibitors, patients positive for aPL, patients treated with anti-Xa agents, and patients with DIC; these values tended to be significantly higher in pregnant women. The height of the first DC was significantly lower in patients who were positive for FVIII inhibitors and was significantly higher in patients treated with anti-Xa agents and pregnant women. The height of the first and second DC was useful for the analysis of hemophilia, FVIII inhibitor, and aPL.

Background Measurement of edoxaban plasma concentration has been gathering attention in major orthopedic surgery patients receiving edoxaban for the prevention of venous thromboembolism (VTE). Methods The anti-Xa activity was measured one hour after edoxaban intake using 3 different assays in 200 patients after major orthopedic surgery. Results The anti-Xa activities on Day 8 were significantly higher than those on Day 4 and those on Day 4 were significantly higher than those on Day 1. The anti-Xa activities in two assays closely correlated with each other, but the other anti-Xa assay did not correlated with other two assays. The anti-Xa activities as detected in the three Xa assays were significantly higher in the patients without deep vein thrombosis (DVT) than in those with DVT on Day 4. Additionally, there were no significant differences in the anti-Xa activities of assays A, B and C between patients with and without massive bleeding (MB) on Days 1, 4, 8 and 15. Conclusion The results of this study suggest that anti-Xa level could be predictive of the risk of VTE, but not of the risk of massive bleeding.

The cutoff values of D-dimer levels for diagnosing or predicting the occurrence of deep vein thrombosis (DVT) were evaluated in preoperative patients and compared to those in non-DVT patients. The levels of 8 different D-dimers were measured using the latex agglutination method or enzyme immunoassay before surgery in 262 orthopedic surgery patients to diagnose subclinical DVT or predict postoperative DVT. There were 15 patients with subclinical DVT and 47 with postoperative DVT, but 200 patients had no DVT at all. All 8 plasma D-dimer values were significantly higher in the patients with subclinical or postoperative DVT than in those without DVT or in healthy volunteers. There were differences in the cutoff values between the assays highly sensitive for low D-dimer levels and wild-range assays of D-dimers. Some D-dimer assays might therefore be more useful than others for diagnosing low levels of D-dimer. Although the measurement of D-dimer levels was useful for diagnosing subclinical DVT and predicting the risk of DVT, the cutoff values for the diagnosis or prediction of DVT varied. The cutoff values for the prediction of postoperative DVT were ≥1.7 µg/mL (D-dimer A-D) and ≥1.0 µg/mL (D-dimer E-H).

Objective Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The early diagnosis of PCD is important for the prevention of long-term sequelae; however, this is often challenging because of the phenotypic heterogeneity of PCD and difficulty in genetic analysis. The majority of PCD patients in Japan are not diagnosed properly. To diagnose PCD more accurately, we developed a targeted next-generation sequencing (NGS) panel. Methods We examined 46 patients (age range, 1–64 years; 23 male and 23 female) who were clinically suspected of PCD. First, mutation hotspots in DNAH5 and DNAI1 were sequenced by the Sanger method. Next, exome sequencing was performed in 32 known PCD genes using our novel NGS panel with the Ion Torrent PGM system. Variant annotation was generated by Ion Reporter Version 5.0 (Life Technologies). Mutations found in the panel were validated by Sanger sequencing. Results Disease-causing gene mutations were found in 10 patients from 7 families: DNAH5 in 4 families, and DNAI1, CCDC40, and RSPH4A in 1 family each. Heterozygous mutations were found in 1 patient. The majority of the mutations found in the present analysis were novel. Conclusion Japanese PCD patients have novel mutations in cilia-related genes. This targeted NGS panel can identify disease-causing mutations in patients with PCD.