Tamao Shinohara

Key Clinical Message: We encountered an extremely low birth weight infant with breast milk‐transmitted cytomegalovirus (CMV) infection. To determine the transmission route, we conducted direct sequence analysis of two variable CMV genes, UL139, and UL146. When utilizing breast milk, the possibility of acquired CMV infection should be considered and tested for prompt diagnosis and treatment.

In patent ductus arteriosus (PDA) in preterm infants, the relationship between treatment timing and long-term developmental prognosis remains unclear. The purpose of this study was to clarify the relationship between the age in days when ductus arteriosus closure occurred and long-term development. Preterm infants with a birth weight of less than 1500 g who were admitted to our NICU over a period of 9 years (2011–2019) and were diagnosed with PDA were included. A new version of the K-type developmental test for corrected ages of 1.5 and 3 years was used as an index of development. The relationship between the duration of PDA and the developmental index was evaluated using Pearson’s correlation coefficient, and multiple regression analysis was performed. Development quotient (DQ) at the ages of 1.5 and 3 years showed a correlation with the PDA closure date and the standard deviation (SD) value of the term birth weight. Multiple regression analysis showed a positive correlation of the DQ at 1.5 and 3 years with the SD value of the term birth weight and a negative correlation with the PDA closure date. In addition, a stronger correlation was found in the “posture/motor” sub-item at 3 years. On the other hand, the analysis including preterm infants without PDA showed that preterm infants with PDA closure on the 6th day or later after birth had a significantly lower 3-year-old DQ than preterm infants with a PDA exposure within 5 days. In conclusion, it is suggested that the decrease in cerebral blood flow due to PDA in preterm infants has an adverse effect on long-term neurodevelopment. Appropriate interventions, including surgical treatment for PDA in preterm infants without delay, ideally within 5 days of birth, may be effective in improving the developmental prognosis.

Acquired cystic lung disease in premature infants is a serious respiratory complication, and pulmonary interstitial emphysema (PIE) has been widely reported. We report a rare case of giant pulmonary bulla in an infant treated with bullectomy where chest computed tomography was useful in directing treatment.

Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B‐cell precursor‐ALL (BCP‐ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA‐incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7‐day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP‐ALL and 23 T‐ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.

Background: Acquired cystic lung disease is a serious respiratory complication of bronchopulmonary dysplasia in premature infants. Most cases of acquired cystic lung disease underlying bronchopulmonary dysplasia involve pulmonary interstitial emphysema. Although this is a reversible condition, there are a few instances where surgery might be necessary. An accurate diagnosis is important to decide the therapeutic strategy for acquired cystic lung disease. Here, we report a rare case of a giant pulmonary bulla in an infant treated with bullectomy. Case presentation: A male infant born at 23 weeks of gestation with a birth weight of 524 g was initially diagnosed with respiratory distress syndrome. During mechanical ventilatory support, he presented with recurrent pneumothorax and a gradually expanding pulmonary cyst in the right lung. Chest CT at 5 months of age revealed a large cyst located in the subpleural area adjacent to the multiple cystic air spaces. These findings are consistent with the diagnosis of giant pulmonary bulla with pulmonary interstitial emphysema underlying bronchopulmonary dysplasia . At 9 months of age, the giant pulmonary bulla expanded further due to acute bronchitis for which he developed respiratory failure and obstructive shock. This warranted a bullectomy for the giant pulmonary bulla. After the operation, the unresected pulmonary interstitial emphysema lesion did not expand further. He is currently three years old and has no respiratory problems. Conclusions: This case demonstrated that chest CT is useful for providing valuable anatomical information necessary in deciding the treatment strategy for acquired cystic lung disease in infants.

Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.

Identification of genetic variants associated with glucocorticoids (GC) sensitivity of leukaemia cells may provide insight into potential drug targets and tailored therapy. In the present study, within 72 leukaemic cell lines derived from Japanese patients with B‐cell precursor acute lymphoblastic leukaemia (ALL), we conducted genome‐wide genotyping of single nucleotide polymorphisms (SNP) and attempted to identify genetic variants associated with GC sensitivity and NR3C1 (GC receptor) gene expression. IC50 measures for prednisolone (Pred) and dexamethasone (Dex) were available using an alamarBlue cell viability assay. IC50 values of Pred showed the strongest association with rs904419 (P = 4.34 × 10⁻⁸), located between the FRMD4B and MITF genes. The median IC50 values of prednisolone for cell lines with rs904419 AA (n = 13), AG (n = 31) and GG (n = 28) genotypes were 0.089, 0.139 and 297 µmol/L, respectively. For dexamethasone sensitivity, suggestive association was observed for SNP rs2306888 (P = 1.43 × 10⁻⁶), a synonymous SNP of the TGFBR3 gene. For NR3C1 gene expression, suggestive association was observed for SNP rs11982167 (P = 6.44 × 10⁻⁸), located in the PLEKHA8 gene. These genetic variants may affect GC sensitivity of ALL cells and may give rise to opportunities in personalized medicine for effective and safe chemotherapy in ALL patients.

T(17;19)(q21‐q22;p13), responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t(1;19)(q23;p13), producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t(17;19)‐ALL is extremely poor, while that of t(1;19)‐ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)‐ALL cases. Using 4 t(17;19)‐ALL and 16 t(1;19)‐ALL cell lines, drug response profiling was analyzed. t(17;19)‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)‐ALL cell lines. Sensitivities to three (Pred, VCR, and l ‐asparaginase [ l ‐Asp]), four (Pred, VCR, l ‐Asp, and DNR) and five (Pred, VCR, l ‐Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)‐ALL cell lines than for t(1;19)‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t(17;19)‐ALL cell lines than in t(1;19)‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t(17;19)‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t(17;19)‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t(17;19)‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t(17;19)‐ALL cell lines. In all four t(17;19)‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)‐ALL.