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Minimal residual disease (MRD) is usually defined as the small number of cancer cells that remain in the body after treatment. The clinical significance of MRD kinetics is well recognized in treatment of hematologic malignancies, particularly acute lymphoblastic leukemia (ALL). Real time quantitative PCR targeting immunoglobulin (Ig) or T-cell receptor (TCR) rearrangement (PCR-MRD), as well as multiparametric flow cytometric analysis targeting antigen expression, are widely used in MRD detection. In this study, we devised an alternative method to detect MRD using droplet digital PCR (ddPCR), targeting somatic single nucleotide variants (SNVs). This ddPCR-based method (ddPCR-MRD) had sensitivity up to 1E-4. We assessed ddPCR-MRD at 26 time points from eight T-ALL patients, and compared it to the results of PCR-MRD. Almost all results were concordant between the two methods, but ddPCR-MRD detected micro-residual disease that was missed by PCR-MRD in one patient. We also measured MRD in stored ovarian tissue of four pediatric cancer patients, and detected 1E-2 of submicroscopic infiltration. Considering the universality of ddPCR-MRD, the methods can be used as a complement for not only ALL, but also other malignant diseases regardless of tumor-specific Ig/TCR or surface antigen patterns.

ETV6-related thrombocytopenia is an autosomal dominant thrombocytopenia, characterized by a bleeding tendency and predisposition to hematological malignancies. The similarity in symptoms makes differentiating immune and congenital thrombocytopenia challenging. We report a 5-year-old girl who presented with chronic thrombocytopenia associated with repetitive and long-lasting epistaxis, leading to blood transfusion for severe anemia. Blood tests showed thrombocytopenia (52 × 103/µL) with normal-sized platelets and transiently low von Willebrand factor (VWF) levels (VWF:RCo 13%, VWF:Ag 50%); therefore, von Willebrand disease type 2 was initially suspected. Repetition of the blood tests revealed normal levels of VWF. Exome and Sanger sequencing identified a germline ETV6 heterozygous variant, c.641C > T:p.(P214L). No additional pathogenic variants were found, including VWF, in the gene panel testing of the 53 known target causative genes for thrombocytopenia. High-throughput exome sequencing for chronic thrombocytopenia can be utilized to differentially diagnose ETV6-related thrombocytopenia from chronic/intractable immune thrombocytopenia and to effectively monitor malignancy.

Antiseizure prophylaxis is required during busulfan administration for hematopoietic stem cell transplantation. However, antiseizure agents such as benzodiazepines and phenytoin may produce adverse effects through interaction with other drugs. We retrospectively assessed the prophylactic efficacy and safety of levetiracetam and clonazepam against busulfan-induced seizures between 2013 and 2018. Thirty patients (after 2015) received levetiracetam, and 13 patients (before 2015) received clonazepam in this study. Levetiracetam was well-tolerated and had a significantly lower frequency of adverse effects, such as somnolence, compared with clonazepam, although two patients in the levetiracetam group experienced seizures. Levetiracetam is a feasible option for preventing busulfan-induced seizures.

The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae), is one of the main insect pests of tea, Camellia sinensis Kuntz, in Japan. Recently, A. honmai has developed a high resistance to diacylhydrazine analog insect growth regulators, such as tebufenozide, in Shizuoka Prefecture. Previously, we identified a point mutation (A415V) in the ecdysone receptor gene (EcR), a candidate factor responsible for tebufenozide resistance. In this study, we have developed a molecular method of diagnosis to detect the EcR A415V mutation by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). This method was confirmed to be successfully applicable to larvae reared in the laboratory and adults collected by pheromone traps in the field. The appearance ratio of the resistant allele in the A. honmai populations from various Japanese districts examined by the method revealed a high correlation with the magnitude of tebufenozide resistance. These results verified that the A415V mutation is the principal factor responsible for tebufenozide resistance and the PCR–RFLP method may be used as a reliable and convenient tool for monitoring tebufenozide resistance in the field.

The smaller tea tortrix, Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae), is one of the most common pests that affect the tea plant, Camellia sinensis (L.) O. Kuntze. Diacylhydrazine-analog insect growth regulators, such as tebufenozide, have been used as controls; however, the species began showing resistance to this compound in approximately 2004. Previously, we identified an amino acid mutation (A415V) in the ecdysone receptor gene (EcR) as a principal factor in tebufenozide resistance and developed a molecular diagnostic method to detect the EcR A415V mutation through polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). However, for the continuous management of tebufenozide resistance in A. honmai, a simple and quick genetic diagnostic method that does not require specialized equipment is preferable. In this study, we developed a novel technique to detect the mutation using allele-specific loop-mediated isothermal amplification (AS-LAMP). This method enables the monitoring and detection of tebufenozide-resistance alleles much faster than PCR–RFLP and with moderate accuracy. Therefore, it could be a powerful tool for the early detection of tebufenozide-resistant A. honmai in tea plantations.

Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that leads to recurrent infection and hyper-inflammation, occasionally represented by CGD-associated colitis (CGD colitis). Although clinical symptoms of CGD colitis mimic those of ulcerative colitis (UC), there is no reliable standard measurement of disease activity or standard therapeutic strategy for CGD colitis. Here, we examined the clinical manifestation of CGD colitis based on severity using a noninvasive measure of disease activity, the Pediatric Ulcerative Colitis Activity Index (PUCAI), which has been validated and widely used for pediatric UC.Sixteen of 35 CGD patients, who were diagnosed with CGD colitis based on colonoscopic and histological findings, were examined using the PUCAI. Both the PUCAI and the physician global assessment (PGA) tool were retrospectively scored by reviewing medical records.Disease activity defined by PUCAI was correlated with PGA, and increased at diagnosis of CGD colitis, especially in patients who were younger than 6 years of age (very early-onset CGD colitis: VEO-CGD colitis) when diagnosed with CGD colitis. All severe patients had a more progressive form of VEO-CGD colitis. Unlike mild and moderate patients, severe patients required multidrug therapy of corticosteroids and immunomodulator/immunosuppressants, and some were eventually treated with hematopoietic stem cell transplantation.Although the validation of PUCAI in CGD colitis should be considered for future use, our results indicate that noninvasive measures could be effective to measure disease activity and help to determine suitable treatment for CGD colitis. In patients with VEO-CGD colitis, multidrug therapy would need to be considered at an early stage on the basis of disease activity.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. CGD patients also suffer from immune regulatory disorders, such as CGD-associated bowel inflammation with granuloma, which could be caused by excessive inflammation without demonstrable infection. We investigated the clinical manifestation of interstitial lung disease (ILD) resulting from excessive inflammation in X-linked CGD patients. Pulmonary CT images and testing of serum KL-6 levels were performed to assess ILD in the patients. For this study, patients with pulmonary lesions due to demonstrable infections were excluded from among ILD patients. Among 33 CGD patients, four developed ILD; they had increased reticulo-nodular opacities on CT images and elevated serum KL-6 levels. Histopathological examinations revealed multiple homogeneous microgranulomas in the lesions of inflammatory cell infiltration. Mononuclear cells obtained from their pulmonary lesions produced higher amounts of inflammatory cytokines than the peripheral blood mononuclear cells of CGD patients, suggesting that the only infiltrating cells in the pulmonary lesions were activated and produced large amounts of inflammatory cytokines in ILD patients. Interestingly, an anti-inflammatory drug, such as a corticosteroid or thalidomide, but not anti-bacterial or anti-fungal drugs, improved CT image findings and reduced their KL-6 levels. CGD patients’ daily exposures to inhaled antigens may induce excessive reactions with the production of inflammatory cytokines leading to the development of ILD with multiple microgranulomas, which could be due to an inadequate production of reactive oxygen species in CGD.

We investigated the susceptibility of the smaller tea tortrix, Adoxophyes honmai Yasuda, to diamide insecticides in the Shimada-Yui tea fields in Shizuoka Prefecture, Japan, from 2006 to 2011. By 2011, the insects had developed significant resistance even to concentrations far above the registration concentrations of two diamides, flubendiamide and chlorantraniliprole. The lethal concentration 50 (LC50) values of flubendiamide showed a rapid annual increase from 16.2 ppm in 2007 to 161 ppm in August 2011, exceeding the registration concentration of 100 ppm in 2010 and 2011. The LC50 values of chlorantraniliprole increased sharply from 25.3 ppm in 2010 to 98.8 ppm in August 2011, exceeding the registration concentration of 50 ppm. The LC50 values for flubendiamide and chlorantraniliprole at 10 days after treatment in insects collected in August 2011 were 105-fold and 77.2-fold higher, respectively, than those in a susceptible strain.

Mutations in genes critical for surfactant metabolism, including surfactant protein C (SP-C) and ABCA3, are well-recognized causes of interstitial lung disease. Recessive mutations in ABCA3 were first attributed to fatal respiratory failure in full-term neonates, but they are also increasingly being recognized as a cause of respiratory disorders with less severe phenotypes in older children and also adults. Here, we report a 20-month-old boy with interstitial lung disease caused by two distinct ABCA3 mutations. Initial treatment with methylprednisolone was unsuccessful, but the additional administration of hydroxychloroquine was effective. The family history revealed that the patient’s older brother had died of idiopathic interstitial lung disease at 6 months of age, suggesting a genetic etiology of the disease. Sequence analyses of SP-C and ABCA3 genes were performed using DNA samples from the patient himself, his parents, and his brother. These analyses revealed novel compound heterozygous mutations in the coding exons of ABCA3 in both the patient and his brother: c.2741A > G, of paternal origin, and c.3715_3716insGGGGGG, of maternal origin. Conclusion Since ABCA3 mutations seem to be a heterogeneous entity with various phenotypes, we recommend genetic testing for mutations in SP-C and ABCA3 genes to be considered in children with unexplained interstitial lung disease.

Although it has been suggested that defective interleukin 7 receptor (IL-7R) signaling is one of the principal causes of severe combined immunodeficiency disease (SCID) in mice and humans, little is known about the molecular and clinical characteristics of human IL-7Rα mutations. We report a novel mutation of the IL-7Rα gene in a Korean SCID patient with a greatly diminished T-cell count but normal numbers of B-cells and natural killer (NK) cells. Using direct sequencing and restriction fragment length polymorphism analysis, we identified a C →T nucleotide change at position 638. This change resulted in a nonsense mutation (R206stop) in this patient. Both parents were heterozygous for C/T at this site. The results of this study emphasize the importance of characterization of IL-7Rα mutations in SCID patients with diminished T-cell numbers but normal numbers of B-cells and NK cells.