TBI, etoposide, and cyclophosphamide conditioning for intermediate-risk relapsed childhood acute lymphoblastic leukemia.

BACKGROUND: In children with intermediate-risk relapsed acute lymphoblastic leukemia (ALL), allogeneic hematopoietic stem cell transplantation (allo-HSCT) has markedly improved the outcome of patients with an unsatisfactory minimal residual disease (MRD) response. Total body irradiation (TBI), etoposide (ETP), and cyclophosphamide (CY) have been shown to be equivalent to or better than TBI + ETP for conditioning, so we hypothesized that even greater survival could be achieved due to recent advances in HSCT and supportive care. PROCEDURE: We prospectively analyzed the efficacy and safety of allo-HSCT with a unified conditioning regimen of TBI + ETP + CY in children with intermediate-risk relapsed ALL, based on MRD in the bone marrow after induction, from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALL-R08-II nationwide cohort (UMIN000002025). RESULTS: Twenty patients with post-induction MRD ≥ 10-3 and two not evaluated for MRD underwent allo-HSCT. Engraftment was confirmed in all patients, and no transplantation-related mortality was observed. The 3-year event-free survival and overall survival rates after transplantation were 86.4% ± 7.3% and 95.5% ± 4.4%, respectively. CONCLUSION: Allo-HSCT based on post-induction MRD with TBI + ETP + CY conditioning was feasible in Japanese children with intermediate-risk relapsed ALL.

Changes in the Incidence of Infantile Spinal Muscular Atrophy in Shikoku, Japan between 2011 and 2020.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. Al-though there was no cure for SMA, newly developed therapeutic drugs (nusinersen, onasemnogene abeparvovec, and risdiplam) have been proven effective for the improvement of motor function and prevention of respiratory insufficiency of infants with SMA. Nusinersen was introduced in Japan in 2017 and onasemnogene abeparvovec in 2020. We hypothesized that the introduction of these drugs might influence the incidence of SMA (more precisely, increase the diagnosis rate of SMA) in Japan. To test this hypothesis, we conducted a second epidemiological study of infantile SMA using questionnaires in Shikoku, Japan between October 2021 and February 2022. The incidence of infantile SMA during the period 2016-2020 was 7.08 (95% confidence interval [CI] 2.45-11.71) per 100,000 live births. According to our previous epidemiological study, the incidence of infantile SMA during 2011-2015 was 2.70 (95% CI 0.05-5.35) per 100,000 live births. The increased incidence of infantile SMA suggests that the widespread news in Japan regarding the introduction of therapeutic agents, nusinersen and onasemnogene abeparvovec, raised clinicians' awareness about SMA, leading to increased and earlier diagnosis of SMA in Shikoku.

PAX5 alterations in an infant case of KMT2A-rearranged leukemia with lineage switch.

Lineage switch is a rare event at leukemic relapse. While mostly known to occur in KMT2A-rearranged infant leukemia, the underlying mechanism is yet to be depicted. This case report describes a female infant who achieved remission of KMT2A-MLLT3-rearranged acute monocytic leukemia, but 6 months thereafter, relapsed as KMT2A-MLLT3-rearranged acute lymphocytic leukemia. Whole exome sequencing of the bone marrow obtained pre-post lineage switch revealed two somatic mutations of PAX5 in the relapse sample. These two PAX5 alterations were suggested to be loss of function, thus to have played the driver role in the lineage switch from acute monocytic leukemia to acute lymphocytic leukemia.

Panel-based next-generation sequencing facilitates the characterization of childhood acute myeloid leukemia in clinical settings.

Acute myeloid leukemia (AML) accounts for ~20% of pediatric leukemia cases. The prognosis of pediatric AML has been improved in recent decades, but it trails that of most other types of pediatric cancer, with mortality rates of 30-40%. Consequently, newer more targeted drugs are required for incorporation into treatment plans. These newer drugs selectively target AML cells with specific gene alterations. However, there are significant differences in genetic alterations between adult and pediatric patients with AML. In the present study, inexpensive and rapid next-generation sequencing (NGS) of >150 cancer-related genes was performed for matched diagnostic, remission and relapse (if any) samples from 27 pediatric patients with AML. In this analysis, seven genes were recurrently mutated. KRAS was mutated in seven patients, NRAS was mutated in three patients, and KIT, GATA1, WT1, PTPN11, JAK3 and FLT3 were each mutated in two patients. Among patients with relapsed AML, six harbored KRAS mutations at diagnosis; however, four of these patients lost these mutations at relapse. Additionally, two genetic alterations (FLT3-ITD and TP53 alterations) were detected among patients who eventually relapsed, and these mutations are reported to be adverse prognostic factors for adult patients with AML. This panel-based, targeted sequencing approach may be useful in determining the genetic background of pediatric AML and improving the prediction of treatment response and detection of potentially targetable gene alterations. RAS pathway mutations were highly unstable at relapse; therefore, these mutations should be chosen as a target with caution. Incorporating this panel-based NGS approach into the clinical setting may allow for a patient-oriented strategy of precision treatment for childhood AML.

Survey of patients with spinal muscular atrophy on the island of Shikoku, Japan.

BACKGROUND: Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder associated with spinal motor neuron loss and characterized by generalized muscle weakness. Only a few reports exist on SMA epidemiology in Japan. Additionally, nusinersen recently became available as a treatment for this condition. We estimated the prevalence of each type of SMA on Shikoku, Japan's fourth-largest major island. METHODS: We sent a questionnaire to all 131 hospitals in Shikoku that have pediatrics or neurology departments from March to September 2019, asking whether each hospital had SMA patients at that time. If so, we sent a second questionnaire to obtain more detailed information on the clinical data and treatment of each patient. RESULTS: A total of 117 hospitals (89.3%) responded to our first questionnaire, and 21 SMA patients were reported, 16 of whom had homozygous deletion of SMN1. Of the 21, nine had SMA type 1, five were type 2, five were type 3, one was type 4, and one was unidentified. The estimated prevalence for all instances of SMA and 5q-SMA was 0.56 and 0.43 per 100,000 people, respectively. Thirteen patients had received nusinersen therapy. Its outcomes varied from no obvious effects and being unable to sit to being able to sit independently. CONCLUSION: Our data showed the prevalence of SMA types 2 and 3 was relatively low on Shikoku compared with previous reports from other countries, suggesting delayed diagnosis may affect the results. Remaining motor function may be one predicting factor. Greater awareness of SMA among clinicians and patients seems necessary for more accurate epidemiological studies.

A deep intronic mutation of c.1166-285 T > G in SLC46A1 is shared by four unrelated Japanese patients with hereditary folate malabsorption (HFM).

Hereditary folate malabsorption (HFM) is an autosomal recessive disease caused by mutations in SLC46A1 encoding the proton-coupled folate transporter (PCFT). HFM patients present with various clinical features including megaloblastic anemia, thrombocytopenia, combined immunodeficiency and neurodevelopmental disorders. In this study, we report the same deep intronic mutation of c.1166-285 T > G shared by four unrelated Japanese patients with HFM. This mutation was shown to generate a cryptic splice donor site for a 168-bp insertion of intron 3 sequences, leading to premature termination in the middle of this insertion. This mutation could be a founder mutation in the Japanese population, but also could be a hot-spot and could be present in undiagnosed HFM patients worldwide because of the difficulty to detect this mutation.

Barth Syndrome: Different Approaches to Diagnosis.

The diagnosis of Barth syndrome is challenging owing to the wide phenotypic spectrum with allelic heterogeneity. Here we report 3 cases of Barth syndrome with phenotypic and allelic heterogeneity that were diagnosed by different approaches, including whole exome sequencing and final confirmation by reverse-transcription polymease chain reaction.

Pediatric intestinal Behçet disease complicated by myeloid malignancies.

Behçet disease (BD) is rarely seen in children. Its clinical manifestations are believed to differ between pediatric and adult patients. The characteristics of BD complicated by myelodysplastic syndrome (MDS) are well established for adult patients; however, because only a few cases of pediatric-onset BD complicated by MDS have been reported, its clinical characteristics remain unknown. We here retrospectively review pediatric-onset BD complicated by myeloid malignancies in Japan, having identified five such patients. All patients were female and had gastrointestinal involvements, but lacked both major features of BD, i.e., uveitis and association with HLA-B51. All patients had advanced MDS or acute myeloid leukemia and received chemotherapy followed by hematopoietic stem cell transplantation. These five cases suggest that intestinal BD and myeloid malignancies have one or more pathophysiological mechanisms in common.

Verification of risk scores to predict i.v. immunoglobulin resistance in incomplete Kawasaki disease.

BACKGROUND: A recent study indicated the efficacy of the addition of prednisolone to i.v. immunoglobulin (IVIG) as initial treatment in patients with higher risk of IVIG resistance. Several different risk scores for predicting IVIG resistance have been proposed, mainly based on typical Kawasaki disease (KD) patients. We investigated the utility of the risk scores to predict IVIG resistance in incomplete KD. METHODS: Clinical records of incomplete KD patients who received a single dose of IVIG between 2005 and 2012 at Kochi Health Sciences Center were retrospectively reviewed. Patients were classified into an IVIG-responsive group and an IVIG-resistant group. The Kobayashi, Egami, and Sano risk scores were calculated for each patient and the proportion of high-risk patients was compared between the two groups for each risk score. RESULTS: For 51 incomplete KD patients, Kobayashi (66.7% vs 47.6%, P = 0.253), Egami (55.6% vs 38.1%, P = 0.274), and Sano (57.1% vs 10.8%, P = 0.068) risk scores identified a higher proportion of high-risk patients in the IVIG-resistant group compared with the IVIG-responsive group, but significant difference was not observed. Sano risk score had the highest OR (6.19; 95%CI: 1.00-38.26). CONCLUSIONS: The proportion of patients identified as being at high risk for IVIG resistance using the Kobayashi, Egami, and Sano risk scores, respectively, was not significantly different between the IVIG-responsive group and the IVIG-resistant group for incomplete KD. Among the three risk scores, the Sano risk score has the best ability to predict IVIG resistance in incomplete KD.

Persistent clonal chromosomal abnormalities in a chronic myeloid leukemia patient.

Clonal cytogenetic abnormalities (CCA) in Philadelphia chromosome (Ph)-negative cells have been reported in a small population of adult chronic myelogenous leukemia (CML) patients during the clinical course, but CCA in pediatric CML patients are rarely reported. We herein report the case of an 8-year-old boy from the onset of CML. Although he had relapse after unrelated bone marrow transplantation when 9 years old, he has since been in complete molecular response on imatinib mesylate treatment. Surprisingly, various CCA have been observed in this patient, including several reciprocal chromosomal translocations in Ph-negative cells for >12 years. Although dysplasia in the bone marrow cells was identified, no overt transformation to myelodysplastic syndrome or acute myeloid leukemia has been observed. The cause of the CCA remains unknown in this patient, and careful observation is required.

Changes in the features of invasive pneumococcal disease after introduction of the seven-valent pneumococcal conjugate vaccine in a regional core hospital of Kochi, Japan.

Since the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in 2007, invasive pneumococcal disease has declined, but the incidence of Streptococcus pneumoniae serotype 19A has risen worldwide. The present study examined changes in the features of invasive pneumococcal disease since the introduction of the PCV7 in Kochi, Japan. Pediatric cases of invasive pneumococcal disease were investigated before and after vaccine introduction (January 2008 to December 2013). Cases of invasive pneumococcal disease tended to decrease after PCV7 introduction. In addition, before introduction of the vaccine, most serotypes causing invasive pneumococcal disease were those included in the vaccine. However, after the introduction, we found cases infected by serotypes not covered by vaccine. Penicillin-resistant S. pneumoniae was the predominant serotype causing invasive pneumococcal disease before introduction of the PCV7, and the susceptibility of this serotype to antibiotics improved after vaccine introduction. Serotype isolates identified after vaccine introduction were also relatively susceptible to antibiotic therapy, but decreased susceptibility is expected.

Ventriculoperitoneal shunt outcomes among infants.

Ventriculoperitoneal shunts (VPSs) are used for the treatment of hydrocephalus. Here we analyzed the outcomes of VPS placements in 24 infants to determine the risk factors for shunt failure. The infants had undergone the initial VPS operation in our hospital between March 2005 and December 2013. They were observed until the end of January 2014. We obtained Kaplan-Meier curves and performed a multivariate Cox regression analysis of shunt failure. Of the 24 cases, the median (range) values for gestational age, birth weight, and birth head circumference (HC) were 37 (27-39) wks, 2,736 (686-3,788) g, and 35.3 (23.0-45.3) cm, respectively. The total number of shunt procedures was 45. Shunt failure rates were 0.51/shunt and 0.0053/shunt/year. Shunt infection rates were 0.13/shunt and 0.0014/shunt/year. The Kaplan-Meier analysis revealed an increased risk for shunt failure in infants <1 month old or in the HC >90%tile. The Cox regression analysis yielded hazard ratios (HRs) of 2.93(95% confidence interval (CI), 0.96-10.95, p=0.059) for age <1 month, and 4.46 (95%CI:1.20-28.91, p=0.023) for the HC >90%tile. The multivariate Cox regression analysis showed adjusted HRs of 17.56 (95%CI:2.69-202.8, p=0.001) for age <1 month, and 2.95 (95%CI:0.52-24.84, p=0.228) for the HC >90%tile. Our findings thus revealed that the risk factors for shunt failure in infants include age <1 month at the initial VPS placement.

Long-term efficacy of bevacizumab and irinotecan in recurrent pediatric glioblastoma.

A 5-year-old boy with glioblastoma relapsed soon after postoperative irradiation in combination with temozolomide. Second-line chemotherapy was also ineffective; therefore, the bevacizumab and irinotecan were given after a third gross-total resection of the tumor. Treatment was interrupted for 1 month due to development of posterior reversible encephalopathy syndrome, but was re-initiated at a lower dose of bevacizumab with prolonged intervals between treatments. The patient was alive and disease free 2 years after initial diagnosis. Bevacizumab and irinotecan are a promising regimen for pediatric cases of recurrent glioblastoma after gross-total resection, although the optimal treatment schedule must be determined on a patient-by-patient basis.

ABL kinase mutation and relapse in 4 pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia cases.

The tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL), which had showed poor prognosis before the dawn of IM treatment. However, if Ph-ALL patients showed IM resistance due to ABL kinase mutation, second-generation TKI, dasatinib or nilotinib, was recommended. We treated 4 pediatric Ph-ALL patients with both IM and bone marrow transplantation (BMT); however, 3 relapsed. We retrospectively examined the existence of ABL kinase mutation using PCR and direct sequencing methods, but there was no such mutation in all 4 diagnostic samples. Interestingly, two relapsed samples from patients who were not treated with IM before relapse did not show ABL kinase mutation and IM was still effective even after relapse. On the other hand, one patient who showed resistance to 3 TKI acquired dual ABL kinase mutations, F359C at the IM-resistant phase and F317I at the dasatinib-resistant phase, simultaneously. In summary, Ph-ALL patients relapsed with or without ABL kinase mutation. Furthermore, ABL kinase mutation was only found after IM treatment, so an IM-resistant clone might have been selected during the IM treatment and intensive chemotherapy. The appropriate combination of TKI and BMT must be discussed to cure Ph-ALL patients.

Needle tract implantation of hepatoblastoma after percutaneous needle biopsy: report of a case.

A 13-year-old boy was referred to us for investigation of a giant liver mass, approximately 16 cm in diameter. Sonographically guided percutaneous needle biopsy was performed and histological examination revealed a fetal-type hepatoblastoma. After four courses of chemotherapy, we performed a left hepatic trisegmentectomy. Follow-up computed tomography, 55 months after the surgery, showed a 1-cm tumor on the route of the preoperative needle biopsy. A second laparotomy revealed a peritonealised tumor, which was excised. The histology of this tumor was identical to that of the primary hepatoblastoma. To our knowledge, this is only the second report of needle tract implantation of hepatoblastoma after percutaneous needle biopsy.

Reliability of a rapid test for the clinical diagnosis of influenza A/H1N1 2009.

BACKGROUND: The rapid diagnosis of a pandemic influenza A/H1N1 2009 (H1N1pdm) virus infection is required in ambulatory care settings, since early identification can prevent further transmission. However, the sensitivity of rapid influenza diagnostic tests (RIDTs) is still questionable, and specific indicators for H1N1pdm and/or false-negative results by RIDTs have not been clearly determined. METHODS: From June to December 2009, nasal swabs from 324 patients at Kochi Health Science Center were used for the diagnosis of infection by RIDT and reverse transcription polymerase chain reaction. RESULTS: The sensitivity of the RIDT was determined to be 80.0% and the specificity 97.1%. Multivariate analysis revealed that the frequencies of contagiousness and headache were significant in patients with H1N1pdm infection, in addition to common symptoms of respiratory infection. These data indicated that the H1N1pdm virus had high infectivity and was harmful to the endocranial environment. In the false-negative group, the time interval between onset and consultation was 5.5 ± 6.5 h (median ± interquartile range), which was significantly shorter than the 11.5 ± 7.0 h in the true-positive group. The sensitivity of the RIDT was significantly low during the time-period within 3 h from onset (56.0%); however after 4 h the sensitivity was determined to be >80%. These data indicated that the concentration of the virus in nasal swabs was elevated over the course of the disease. CONCLUSIONS: We have demonstrated that the RIDT is reliable for the diagnosis of H1N1pdm infection. Taking into consideration the time interval between onset and consultation and other features of H1N1pdm, such as contagiousness and headache, it may be necessary to re-test RIDT-negative cases later.

Comparison of two methods for evaluating bone marrow metastasis of neuroblastoma: Reverse transcription-polymerase chain reaction for tyrosine hydroxylase and magnetic resonance imaging.

BACKGROUND: The presence of bone marrow (BM) metastasis and circulating tumor cells in patients with neuroblastoma is a significant prognostic factor at diagnosis and might antedate detection of a relapse by other diagnostic studies. In this study, the clinical value of reverse transcription-polymerase chain reaction (RT-PCR) to amplify mRNA for tyrosine hydroxylase (TH) and magnetic resonance imaging (MRI) during the clinical course of patients with advanced neuroblastoma, was evaluated. METHODS: Four patients with Stage 1, 4 or 4S neuroblastoma, were studied. BM and peripheral blood (PB), including peripheral blood stem cell (PBSC), samples were examined for TH mRNA using RT-PCR. Concurrently, MRI detection of BM metastasis was used. RESULTS: In all cases, except one that had no evidence of BM invasion, TH mRNA in BM and PB at diagnosis were positive, and TH mRNA at diagnosis disappeared after chemotherapy. In two cases, although involvement in the neurocentrum BM was detected by MRI, TH mRNA in the iliac crest BM was negative. The pathological area still remained on MRI after intensive therapy. CONCLUSION: RT-PCR for TH mRNA might be the most sensitive method for the detection of occult neuroblastoma cells in BM and PB. However, because invasion of the BM by neuroblastoma may have a focal distribution, sampling errors can occur. Therefore, not only RT-PCR but also MRI, need to be used to rule out marrow involvement, especially at diagnosis and BM relapse.

Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia.

Identification of the targets of mixed lineage leukemia (MLL) fusion genes will assist in understanding the biology of MLL fusion gene leukemias and in development of better therapies. Numerous studies have implicated HOXA9 as one of the possible targets of MLL fusion proteins. To determine if HOXA9 was required for leukemia development by MLL fusion genes, we compared the effects of the Mll-AF9 knock-in mutation in mice in the presence or absence of Hoxa9. Both groups of mice showed myeloid expansion at 8 weeks and then developed myeloid leukemia with a similar incidence and time course. The leukemia in the mice lacking Hoxa9 generally displayed a more immature myeloid phenotype than that in the mice that were wild-type for Hoxa9. Gene expression profiling revealed that expression of Mll-AF9 led to overexpression of Hoxa5, Hoxa6, Hoxa7, Hoxa9, and Hoxa10. Thus, genes of the Hox-a cluster are important in defining the phenotype but not the incidence of Mll-AF9 leukemia. These results demonstrate that the Mll-AF9 fusion gene disrupts the expression of several Hox genes, none of which as a single gene is likely to be necessary for development of leukemia. Instead, we propose that the "Hox code" minimally defined by the Hoxa5-a9 cluster is central to MLL leukemogenesis.

Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation.

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.