Pathological Pleural Invasion is a Risk Factor for Late Recurrence in Long-Term Survivors of Non-small Cell Lung Cancer after Complete Resection.

BACKGROUND: Information regarding late recurrence after pulmonary resection for non-small cell lung cancer (NSCLC) is limited. This study aimed to analyze the risk factors for late recurrence after surgery for NSCLC in the current era. PATIENTS AND METHODS: We conducted a retrospective study of patients who underwent complete resection for pathological I-III NSCLC between 2006 and 2015. Late recurrence was defined as a recurrence that met the following conditions: (1) the patient underwent chest computed tomography (CT) at or after 54 months after surgery and recurrence was not detected at that time, and (2) recurrence that occurred more than 5 years after surgery. The factors influencing late recurrence, relapse-free survival (RFS), and overall survival (OS) after surgery were analyzed. RESULTS: A total of 1275 with 5-year relapse-free survival after surgery were enrolled in this study. The mean age of the patients was 66.4 years and 54% of the patients were men. The median interval between surgery and the latest follow-up examination was 98 months. In total, 35 patients (2.7%) experienced late recurrence and 138 patients have died thus far. The cumulative recurrence, RFS, and OS rates at 10 years were 3.9%, 84.9%, and 86.3%, respectively. A multivariate analysis revealed that pleural invasion was an independent risk factor for late recurrence. Pleural invasion was a poor prognostic factor for both RFS and OS. CONCLUSIONS: Pleural invasion was a predictor of late recurrence. Age > 67 years, preoperative serum carcinoembryonic antigen (CEA) > 5 ng/ml, non-adenocarcinoma, and pleural invasion were poor prognostic factors for RFS.

Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse, and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group AML-D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival [28.6% vs. 90.5%, P < 0.001; 25.0% vs. 89.5%, P < 0.001] than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Successful Bone Marrow Transplantation in a Patient with Acute Myeloid Leukemia Developed from Severe Congenital Neutropenia Using Modified Chemotherapy and Conditioning Regimen for Leukemia.

Severe congenital neutropenia (SCN) is characterized by chronic neutropenia with recurrent infections from early infancy and a predisposition to myelodysplastic syndrome/acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with SCN who develop myelodysplastic syndrome/AML. We report an 8-year-old girl with SCN carrying an ELANE mutation that had been refractory to granulocyte colony-stimulating factor. The patient experienced recurrent infections and then developed AML. The counts of leukemic blasts that harbored both CSF3R and RUNX1 mutations spontaneously decreased with antimicrobial therapy, leading to partial remission. After AML recurrence, HSCT was successfully performed using modified chemotherapy and a conditioning regimen. Serial donor lymphocyte infusions against mixed chimerism induced complete donor chimerism over 4 years without any infections or AML relapse. This case suggests the importance of carefully managing neutropenia-related infections, leukemia progression, and HSCT in patients with SCN developing AML.

The impact of multidisciplinary approaches on the outcomes of olfactory neuroblastoma treated with postoperative radiotherapy.

BACKGROUND: We investigated the outcomes of postoperative radiation therapy for olfactory neuroblastoma (ONB) and our cross-departmental collaboration to enhance the effectiveness of cancer treatment. METHODS: We retrospectively evaluated 22 patients with ONB who underwent postoperative radiotherapy after tumor resection. En bloc resection was performed; pathology specimens were prepared in coronal sections; and irradiation fields were determined after discussion with radiation oncologists, head and neck surgeons, and pathologists. RESULTS: The overall survival and local control rates were 95.5% and 100%, respectively, at a median 37-month follow-up. The 3- and 5-year disease-free survival (DFS) rates were 64.4% and 56.3%, respectively. Of the 22 patients, 9 (8 Kadish C and 1 Kadish B) had disease recurrence. Of the nine patients, five had positive margins and two had closed margins; cervical lymph node recurrence occurred in six, and distant metastasis with or without cervical lymph node recurrence occurred in three. DFS analysis of risk factors showed no statistically significant differences, but positive margins were a significant recurrence factor in multivariate analysis. CONCLUSIONS: The local control rate of ONB treated with postoperative radiation therapy was 100%. This may be attributed to cross-departmental cooperation between head and neck surgeons, pathologists, and radiation oncologists, which resulted in accurate matching of CT images for treatment planning with the location of the tumor and positive margins. Longer follow-up periods are required to evaluate the effectiveness of our strategy.

Factors Associated with Overlap between Functional Dyspepsia and Nonerosive Reflux Disease in Endoscopy-based Helicobacter pylori-uninfected Japanese Health Checkup Participants: A Prospective, Multicenter Cross-sectional Study.

Objective We assessed the factors associated with overlap between functional dyspepsia (FD) and nonerosive reflux disease (NERD) in endoscopy-based Helicobacter pylori-uninfected Japanese health checkup participants. Methods We utilized baseline data from 3,085 individuals who underwent upper endoscopy for health screening in a prospective, multicenter cohort study. The participants were asked to complete a questionnaire detailing their upper abdominal symptoms and lifestyle. Anxiety was assessed using the State-Trait Anxiety Inventory (STAI) score. FD, postprandial distress syndrome (PDS), and epigastric pain syndrome (EPS) were defined according to the Rome III criteria. NERD was defined as heartburn or regurgitation ≥1 day/week without erosive esophagitis. Results Of the 3,085 participants, 73 (2.4%), 97 (3.1%), and 84 (2.7%) had FD alone, NERD alone, and FD-NERD overlap, respectively. Factors associated with FD-NERD-overlap participants compared with participants with neither FD nor NERD were women [odds ratio (OR): 2.08, 95% confidence interval (CI): 1.24-3.52], body mass index (BMI) <18.5 (OR: 2.87, 95% CI: 1.56-5.07), alcohol consumption ≥20 g/day (OR: 1.85, 95% CI: 1.06-3.15), and a high STAI score (OR: 2.53, 95% CI: 1.62-4.00). Increasing age (OR: 1.06, 95% CI: 1.01-1.11) and EPS symptoms [pure EPS (OR: 3.67, 95% CI: 1.65-8.51) and PDS-EPS overlap (OR: 11.6, 95% CI: 4.09-37.2)] were associated with FD-NERD overlap vs. FD alone. Women (OR: 3.17, 95% CI: 1.47-7.04), BMI <18.5 (OR: 3.03, 95% CI: 1.04-9.90), and acid reflux symptoms ≥2 days a week (OR: 3.57, 95% CI: 1.83-7.14) were associated with FD-NERD overlap vs. NERD alone. Conclusion Understanding the clinical features of overlap between FD and NERD will lead to better management.

Production of recombinant humanized monoclonal anti-human neutrophil antigen (HNA) antibodies with potential applicability as standard antibodies.

BACKGROUND: Antibodies against human neutrophil antigen (HNA) are involved in the pathogenesis of neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. The present methods for anti-HNA antibody identification strongly depend on the presence of standard antisera with known allo/isospecificities. Here, we aimed to produce recombinant humanized antibodies to HNA from available mouse monoclonal antibodies (MoAbs). STUDY DESIGN AND METHODS: RNAs were extracted from available hybridoma cells producing mouse anti-HNA antibodies recognizing HNA-1a (TAG-1), -1b (TAG-2), -2 (TAG-4), and FcγRIIIb, and the cDNA was synthesized. Recombinant fragments consisting of the variable regions of the H and L chains of the mouse MoAb ligated to the constant region of human IgG were incorporated into an expression vector and transfected into CHO cells. Antibody specificity of the selected humanized monoclonal antibodies was confirmed, and tested by the participants of the ISBT Granulocyte Immunobiology Working Party (GIWP) workshop 2020. RESULTS: GIFT results confirmed the specific reactivity of TAGH-1 to -4, except for a cross-reactivity of TAGH-2 with HNA-1a/a neutrophils, only in flow-cytometry. MAIGA results showed clear specificity of all humanized antibodies, but the selection of the appropriate capture monoclonal antibody was essential for the test. The results of the ISBT GIWP showed high concordance among the labs. CONCLUSIONS: These are the first humanized monoclonal antibodies to HNA-1 and HNA-2 antigens produced and they will be important standard reagents for laboratories testing for neutrophil antibodies. We plan to have these humanized MoAbs available through WHO.

Clinical significance of human neutrophil antigen-1 antibodies in children with neutropenia.

Primary autoimmune neutropenia in young children is characterized by chronic neutropenia and positivity for antibodies against human neutrophil antigens (HNAs). This study analyzed the clinical characteristics of 402 children with neutropenia to identify differences between those with and without HNA-1 antibodies (HNA1abs). HNAabs in sera were detected by granulocyte immunofluorescence testing using flow cytometry. Relative fluorescence intensity (RFI) values were used to divide patients into positive (PG, n = 302), borderline (BG, n = 34), and negative (NG, n = 66) groups. The antibodies reacted to HNA-1a alone (59%), HNA-1b alone (1%), and HNA-1a/1b (40%). The PG had a significantly lower absolute neutrophil count before definitive diagnosis and a 1.6- to 2-times greater risk of hospitalization during neutropenia than the other groups. The median duration of neutropenia was longest in the PG at 25 months, followed by 20 months in the BG and 14 months in the NG. This large-scale cohort characterizes clinically distinct groups using the RFI value for HNA1abs in young children with neutropenia. Detection of HNA1abs may aid in understanding the clinical characteristics of children with neutropenia.

A case of alpha-fetoprotein-positive thymic small cell carcinoma: a case report.

BACKGROUND: An alpha-fetoprotein (AFP)-positive neuroendocrine tumor of the thymus is a rare thoracic malignancy. Few cases of AFP-positive thymic large cell neuroendocrine carcinoma have been reported, with no known previous report of an AFP-positive thymic small cell carcinoma. We encountered a patient with an AFP-positive small cell carcinoma and report here the clinical course. CASE PRESENTATION: A 40-year-old man was transferred to our hospital for a large anterior mediastinal tumor and showed an elevated serum AFP level. Computed tomography-guided biopsy results led to diagnosis of small cell carcinoma. Induction chemoradiotherapy was performed before surgery because of pulmonary artery invasion. The response to Induction chemoradiotherapy varied among sites, with the main tumor showing shrinkage and the metastasis site growth. This discrepancy suggested a histologic type unresponsive to or cancer cells potentially resistant to chemotherapy, thus a surgical re-biopsy was performed and histological findings revealed AFP-positive small cell carcinoma. Additional chemotherapy was performed, though could not control cancer progression, and the patient died 8 months after the first medical examination. CONCLUSIONS: Our present clinical experience indicates the importance of histological examination for determining AFP-positive anterior mediastinal tumor treatment. Although AFP-positive neuroendocrine tumor of the thymus is relatively rarer than germ cell carcinoma, differential diagnosis with use of a histological examination should be considered because of the potentially poorer prognosis. The present clinical findings for an AFP-positive neuroendocrine tumor of the thymus case are considered useful for establishing an optimal treatment strategy in the future.

Age-specific incidence of joint disease in paediatric patients with haemophilia: A single-centre real-world outcome based on consecutive US examination.

INTRODUCTION: Joint health is one of the most important factors contributing to a healthy life in patients with haemophilia. Recent study revealed that starting early prophylaxis was not enough to prevent joint disease in most paediatric patients with haemophilia. AIM: In this study, we aimed to determine the age-specific incidence of acute joint disease during childhood at single haemophilia treatment centre (HTC). METHOD: The joint health in 48 patients was evaluated based on consecutive US testing for 5 years at annual multidisciplinary comprehensive care. RESULTS: During the study period, 23 patients (47.9%) had no joint disease since the initial examination, whereas 13 patients (27.0%) showed development from negative to positive findings. The incidence of joint disease increased with age: 0% in preschool, 5.3% in elementary school, 14.3% in junior high school and 35% beyond high school age. Among the 13 patients who developed joint disease, two experienced acquired synovitis that resolved during the follow-up period. Statistical analysis revealed that the patients who routinely underwent follow-up by the HTC exhibited a significantly lower incidence of joint disease than did those followed up at other institutions (p < .001). CONCLUSION: These results indicated that close check-up, including routine joint examination using US as well as frequent assessment of pharmacokinetic profile at the HTC, might play an important role in avoiding joint disease among paediatric patients with haemophilia.

Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

Clinical conditions and risk factors for inhibitor-development in patients with haemophilia: A decade-long prospective cohort study in Japan, J-HIS2 (Japan Hemophilia Inhibitor Study 2).

BACKGROUND: Inhibitor-development is a serious complication in patients with haemophilia (PwH). Previous studies reported that therapeutic and genetic factors could be associated with these alloantibodies. Relevant clinical features such as genetic-background and different treatment regimens in Japan remain unclear, however. AIMS: To analyse a nation-wide Japanese registry for PwH, and to examine risk factors for inhibitor-development. METHODS AND RESULTS: Newly diagnosed patients with haemophilia A (PwHA) or haemophilia B (PwHB) without inhibitors after 2007, and with treatment records traceable from 0 to 75 exposure days (ED), were enrolled in the Japan Hemophilia Inhibitor Study 2 (J-HIS2) initiated in 2008. Of 417 patients (340 PwHA, 77 PwHB) from 46 facilities, 83 (76 PwHA, 7 PwHB) were recorded with inhibitors by July 2020. Inhibitors were observed in 31.0% of severe PwHA, 8.0% moderate and 1.6% mild and in 17.1% of severe PwHB. The majority of inhibitors (89.7% in severe PwHA and 71.4% in severe PwHB) were detected on or before 25ED (median 12ED in PwHA and 19ED in PwHB). Genotyping in these severe patients identified an association between inhibitor-development and null variants of F8 (P < .01) or F9 (P < .05). A lower incidence of inhibitors was recorded in severe PwHA treated with prophylaxis than in those treated on-demand (P < .01). A past-history of intracranial-haemorrhage appeared to be associated with inhibitor-development, while FVIII-concentrates infusion and routine vaccination on the same day was not related to inhibitor-development. CONCLUSION: The J-HIS2 study has identified significant clinical variables associated with inhibitor-development in Japanese PwH, consistent with other global studies.

Medical checkup data analysis method based on LiNGAM and its application to nonalcoholic fatty liver disease.

Although medical checkup data would be useful for identifying unknown factors of disease progression, a causal relationship between checkup items should be taken into account for precise analysis. Missing values in medical checkup data must be appropriately imputed because checkup items vary from person to person, and items that have not been tested include missing values. In addition, the patients with target diseases or disorders are small in comparison with the total number of persons recorded in the data, which means medical checkup data is an imbalanced data analysis. We propose a new method for analyzing the causal relationship in medical checkup data to discover disease progression factors based on a linear non-Gaussian acyclic model (LiNGAM), a machine learning technique for causal inference. In the proposed method, specific regression coefficients calculated through LiNGAM were compared to estimate the causal strength of the checkup items on disease progression, which is referred to as LiNGAM-beta. We also propose an analysis framework consisting of LiNGAM-beta, collaborative filtering (CF), and a sampling approach for causal inference of medical checkup data. CF and the sampling approach are useful for missing value imputation and balancing of the data distribution. We applied the proposed analysis framework to medical checkup data for identifying factors of Nonalcoholic fatty liver disease (NAFLD) development. The checkup items related to metabolic syndrome and age showed high causal effects on NAFLD severity. The level of blood urea nitrogen (BUN) would have a negative effect on NAFLD severity. Snoring frequency, which is associated with obstructive sleep apnea, affected NAFLD severity, particularly in the male group. Sleep duration also affected NAFLD severity in persons over fifty years old. These analysis results are consistent with previous reports about the causes of NAFLD; for example, NAFLD and metabolic syndrome are mutual and bi-directionally related, and BUN has a negative effect on NAFLD progression. Thus, our analysis result is plausible. The proposed analysis framework including LiNGAM-beta can be applied to various medical checkup data and will contribute to discovering unknown disease factors.

Prospective validation of the provisional entity of refractory cytopenia of childhood, proposed by the World Health Organization.

In 2008, the World Health Organization proposed a new entity of childhood myelodysplastic syndrome (MDS), which was referred to as refractory cytopenia of childhood (RCC). However, whether this morphological classification reflects clinical outcomes remains unclear. We performed a prospective evaluation of bone marrow morphology in 252 children with acquired bone marrow failure between 2009 and 2013. Of 252 patients, 63 were diagnosed with aplastic anaemia (AA), 131 with RCC without multilineage dysplasia (RCC-w/o-MLD) and 58 with RCC with MLD (RCC-MLD). One patient with AA, three with RCC-w/o-MLD and nine with RCC-MLD presented with chromosomal abnormalities at diagnosis (P = 0·001). The response rates to immunosuppressive therapy (IST) at 6 months and the cumulative incidence of clonal evolution at 5 years did not significantly differ among the three groups. A multivariate analysis revealed that the morphological classification of RCC-MLD was a significant risk factor for secondary graft failure after haematopoietic cell transplantation (HCT) (P = 0·003). In view of these findings, RCC could be divided into two categories, RCC-w/o-MLD and RCC-MLD, because children with this condition exhibited a distinct morphology, frequent chromosomal abnormalities at diagnosis and a high frequency of secondary graft failure after HCT.

Enhanced osteoclastogenesis in patients with MSMD due to impaired response to IFN-γ.

BACKGROUND: Patients with Mendelian susceptibility to mycobacterial disease (MSMD) experience recurrent and/or persistent infectious diseases associated with poorly virulent mycobacteria. Multifocal osteomyelitis is among the representative manifestations of MSMD. The frequency of multifocal osteomyelitis is especially high in patients with MSMD etiologies that impair cellular response to IFN-γ, such as IFN-γR1, IFN-γR2, or STAT1 deficiency. OBJECTIVES: This study sought to characterize the mechanism underlying multifocal osteomyelitis in MSMD. METHODS: GM colonies prepared from bone marrow mononuclear cells from patients with autosomal dominant (AD) IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 gain of function (GOF) and from healthy controls were differentiated into osteoclasts in the presence or absence of IFN-γ. The inhibitory effect of IFN-γ on osteoclastogenesis was investigated by quantitative PCR, immunoblotting, tartrate-resistant acid phosphatase staining, and pit formation assays. RESULTS: Increased osteoclast numbers were identified by examining the histopathology of osteomyelitis in patients with AD IFN-γR1 deficiency or AD STAT1 deficiency. In the presence of receptor activator of nuclear factor kappa-B ligand and M-CSF, GM colonies from patients with AD IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 GOF differentiated into osteoclasts, similar to GM colonies from healthy volunteers. IFN-γ concentration-dependent inhibition of osteoclast formation was impaired in GM colonies from patients with AD IFN-γR1 deficiency or AD STAT1 deficiency, whereas it was enhanced in GM colonies from patients with STAT1 GOF. CONCLUSIONS: Osteoclast differentiation is increased in AD IFN-γR1 deficiency and AD STAT1 deficiency due to an impaired response to IFN-γ, leading to excessive osteoclast proliferation and, by inference, increased bone resorption in infected foci, which may underlie multifocal osteomyelitis.

Epidemiology of child mortality and challenges in child death review in Japan: The Committee on Child Death Review: A Committee Report: The Committee on Child Death Review: A Committee Report.

We performed a retrospective survey and verification of the medical records of death cases of children (and adolescents; aged <18 years) between 2014 and 2016 in pediatric specialty training facilities in Japan. Of the 2,827 registered cases at 163 facilities, 2,348 cases were included. The rate of identified deaths compared with the demographic survey, was 18.2%-21.0% by age group. The breakdown of deaths was determined as follows: 638 cases (27.2%) were due to external factors or unknown causes, 118 (5.0%) were suspected to involve child maltreatment, 932 (39.7%) were of moderate or high preventability or were indeterminable. Further detailed verification was required for 1,333 cases (56.8%). Comparison of the three prefectures with high rates of identified deaths in Japan revealed no significant differences, such as in the distribution of diseases, suggesting that there was little selection bias. The autopsy rate of deaths of unknown cause was 43.4%, indicating a high ratio of forensic autopsies. However, sufficient clinical information was not collected; therefore, thorough evaluations were difficult to perform. Cases with a moderate or high possibility of involvement of child maltreatment accounted for 5%, similar to previous studies. However, more objective evaluation is necessary. Preventable death cases including potentially preventable deaths accounted for 25%, indicating that proposals need to be made for specific preventive measures. Individual primary verification followed by secondary verification by multiple organizations is effective. It is anticipated that a child death review (CDR) system with such a multi-layered structure will be established; however, the following challenges were revealed: The subjects of CDR are all child deaths. Even if natural death cases are entrusted to medical organizations, and complicated cases to other special panels, the numbers are very high. Procedures need to be established to sufficiently verify these cases. Although demographic statistics are useful for identifying all deaths, care must be taken when interpreting such data. Detailed verification of the cause of death will affect the determination of subsequent preventability. Verification based only on clinical information is difficult, so a procedure that collates non-medical information sources should be established. It is necessary to organize the procedures to evaluate the involvement of child maltreatment objectively and raise awareness among practitioners. To propose specific preventive measures, a mechanism to ensure multiprofessional diverse perspectives is crucial, in addition to fostering the foundation of individual practitioners. To implement the proposed measures, it is also necessary to discuss the responsibilities and authority of each organization. Once the CDR system is implemented, verification of the system should be repeated. Efforts to learn from child deaths and prevent deaths that are preventable as much as possible are essential duties of pediatricians. Pediatricians are expected to undertake the identified challenges and promote and lead the implementation of the CDR system. This is a word-for-word translation of the report in J. Jpn. Pediatr. Soc. 2019; 123 (11): 1736-1750, which is available only in the Japanese language.

Osteoblastic adherence regulates hematopoietic stem cell self-renewal and differentiation: a conceptional in vitro and in vivo study.

BACKGROUND: Intrinsic factors related to self-renewal regulatory factors in hematopoietic stem cells are well known; however, limited information is available on extrinsic factors, such as the cell environment. Therefore, in this study, we analyzed the regulatory mechanism of hematopoietic stem cell self-renewal, focusing on the osteoblastic niche, and examined how adherence to osteoblasts affects stem cell differentiation. METHODS: For this experimental study, we developed a co-culture system for hematopoietic stem cells and osteoblasts, such that cells adhered to osteoblasts can be separated from those that do not. Murine Sca1-positive cells were separated into groups according to whether they were attached to osteoblasts or detached from osteoblasts, and each group was then subjected to colony assays and bone marrow transplantation experiments. RESULTS: Adhered Sca1-positive cells developed more secondary colonies than non-adhered Sca1-positive cells. Furthermore, in bone marrow transplantation experiments, adhered Sca1-positive cells showed successful engraftment. We explored the role of Polycomb genes in the regulation of cell fate and found that self-renewing cells attached to osteoblasts had high Bmi-1 expression and low Mel-18 expression, while this expression was reversed in differentiating cells. CONCLUSIONS: Our results suggest that hematopoietic stem cells self-renew when they remain in osteoblastic niches after cell division. Further, when stem cells leave the niches, they undergo differentiation.

NBS1 I171V variant underlies individual differences in chromosomal radiosensitivity within human populations.

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.

Application of Genome-Wide DNA Methylation Analysis to Differentiate a Case of Radiation-Induced Glioblastoma From Late-Relapsed Medulloblastoma.

Recurrent medulloblastoma can be difficult to diagnose with conventional diagnostic methods because other lesions mimic tumor relapse, particularly at later stages. We report 2 cases of medulloblastoma, both of which seemed to develop late recurrences. Case 1 was a 6-year-old girl who had a medulloblastoma with focal desmoplasia. She was in complete remission for 9 years after treatment but developed an intradural lesion in her thoracic spine, which was pathologically confirmed as tumor recurrence by biopsy. Case 2 was a 10-year-old girl who had a nonmetastatic medulloblastoma. She developed a left cerebellar mass 5 years after the initial diagnosis; the pathological diagnosis was tumor relapse. We performed t-distributed stochastic neighbor embedding of the methylation data from these cases and reference data. In contrast to the consistency of methylation profiling and copy number abnormalities between primary and recurrent tumors of Case 1, the analysis of the recurrent tumor in Case 2 was distinct from medulloblastomas and clustered with "IDH-wild type glioblastomas," suggesting that the recurrent tumor was a radiation-induced glioblastoma. This report highlights the clinical utility of molecular genetic/epigenetic analysis combined with a standard diagnostic approach to confirm the diagnosis of brain tumor recurrence.

Inherited CARD9 Deficiency in a Child with Invasive Disease Due to Exophiala dermatitidis and Two Older but Asymptomatic Siblings.

PURPOSE: Autosomal recessive CARD9 deficiency predisposes patients to invasive fungal disease. Candida and Trichophyton species are major causes of fungal disease in these patients. Other CARD9-deficient patients display invasive diseases caused by other fungi, such as Exophiala spp. The clinical penetrance of CARD9 deficiency regarding fungal disease is surprisingly not complete until adulthood, though the age remains unclear. Moreover, the immunological features of genetically confirmed yet asymptomatic individuals with CARD9 deficiency have not been reported. METHODS: Identification of CARD9 mutations by gene panel sequencing and characterization of the cellular phenotype by quantitative PCR, immunoblot, luciferase reporter, and cytometric bead array assays were performed. RESULTS: Gene panel sequencing identified compound heterozygous CARD9 variants, c.1118G>C (p.R373P) and c.586A>G (p.K196E), in a 4-year-old patient with multiple cerebral lesions and systemic lymphadenopathy due to Exophiala dermatitidis. The p.R373P is a known disease-causing variant, whereas the p.K196E is a private variant. Although the patient's siblings, a 10-year-old brother and an 8-year-old sister, were also compound heterozygous, they have been asymptomatic to date. Normal CARD9 mRNA and protein expression were found in the patient's CD14+ monocytes. However, these cells exhibited markedly impaired pro-inflammatory cytokine production in response to fungal stimulation. Monocytes from both asymptomatic siblings displayed the same cellular phenotype. CONCLUSIONS: CARD9 deficiency should be considered in previously healthy patients with invasive Exophiala dermatitidis disease. Asymptomatic relatives of all ages should be tested for CARD9 deficiency. Detecting cellular defects in asymptomatic individuals is useful for diagnosing CARD9 deficiency.

Individual Differences in Autistic Traits are Associated with Serotonin Transporter Gene Polymorphism Through Medial Prefrontal Function: A Study Using NIRS.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder that can vary considerably in severity. Autistic traits are distributed continuously across populations, even in sub-clinical individuals. Serotonin transporter-gene polymorphic region (5-HTTLPR) has been studied as a candidate genetic factor related to ASD, however results have been inconsistent. 5-HTTLPR is implicated in the function of medial prefrontal cortex (mPFC), a region associated with the social abnormalities found in ASD. Here we hypothesize that autistic traits are affected by the 5-HTTLPR genotype indirectly through mPFC mediation. Using near-infrared spectroscopy (NIRS), we first examined mPFC activation in people with ASD when they performed a facial affect-labeling task. Compared with a typical development group, the ASD group showed significantly lower mPFC activation during the task. Using the same task paradigm, we next investigated the relationship between autistic traits and 5-HTTLPR in sub-clinical participants, and whether associations were mediated by mPFC function. Correlation analyses indicated that participants with a large number of 5-HTTLPR L-alleles had high-level autistic traits related to social skills and low right mPFC activation. We also observed a significant negative correlation between autistic traits related to social skills and right mPFC activation. Structural equation analysis suggested a significant indirect effect of 5-HTTLPR on Autism-Spectrum Quotients, with right mPFC activation acting as a mediator. These results suggest that the diverse autistic traits related to social skills seen in the general population are associated with the 5-HTTLPR genotype, and that this association is mediated by right mPFC function.