Alteration of the immune environment in bone marrow from children with recurrent B cell precursor acute lymphoblastic leukemia.

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

Prognostic factors of children and adolescents with T-cell acute lymphoblastic leukemia after allogeneic transplantation.

Acute lymphoblastic leukemia (ALL) is the most common cancer during childhood, and some high-risk patients with ALL require hematopoietic stem cell transplantation (HSCT). Mainly due to small patient numbers, studies focusing specifically on children and adolescents with T-cell ALL (T-ALL) are limited. Using a nationwide registry, we retrospectively analyzed data from patients under 20 years old who underwent their first HSCT for T-ALL between 2000 and 2018. As a result, total 484 patients were included, and their median follow-up period was 6.9 years after HSCT for survivors. While patients receiving HSCT at first complete remission (CR) showed relatively good 5-year leukemia free survival (5yLFS, 73.5%), once relapse occurred, their prognosis was much worse (44.4%) even if they attained second remission again (p < 0.001). Among patients receiving HSCT at CR1, grade II-IV acute graft versus host disease was associated with worse overall and LFS than grade 0-I (5yLFS 69.5% vs. 82.1%, p = 0.026) mainly due to high non-relapse mortality. Among those patients, patients receiving related bone marrow transplantation, unrelated bone marrow transplantation, or unrelated cord blood transplantation showed similar survival (5yLFS, 73.2%, 76.3%, and 77.0%, respectively). For patients undergoing cord blood transplantation at CR1, total-body irradiation-based myeloablative conditioning was associated with better 5yLFS than other conditioning regimens (85.4% vs. 62.2%, p = 0.044), as it reduced the risk of relapse. These results indicate that relapsed patients have much less chance of cure, and that identifying patients who require HSCT for cure and offering them HSCT with optimal settings during CR1 are crucial for children and adolescents with T-ALL.

KMT2A-rearranged diffuse large B-cell lymphoma in a child: a case report and molecular characterization.

KMT2A-rearranged diffuse large B-cell lymphoma (DLBCL) is rare in both the adult and pediatric populations, and its biological features are unclear. We here report the case of a 19-month-old female with a right temporal bone tumor that was ultimately diagnosed as DLBCL by tumor biopsy. There was no morphological evidence of bone marrow infiltration at diagnosis. The tumor nearly completely dissolved after scheduled chemotherapy for mature B-cell lymphoma; however, leukemic conversion occurred 2 months after completion of chemotherapy. Additional chemotherapy including hematopoietic cell transplantation in a non-remission state was unsuccessful, and disease progression ultimately resulted in the death of the patient 18 months after the diagnosis. We detected the KMT2A-MLLT3 fused transcript in the bone marrow of the patient with primary and recurrent cancer. RNA-sequencing of the bone marrow with recurrent cancer confirmed the KMT2A-MLLT3 fusion gene, although fusion genes involving BCL6, BCL2, or were not detected. Moreover, RNA-sequencing revealed overexpression of MEIS1 and MEF2C, which are highly expressed in KMT2A-rearranged leukemia, whereas the HOXA gene cluster was not overexpressed. The current case formed part of the KMT2A-rearranged acute lymphoblastic leukemia cluster in a T-distributed stochastic neighbor embedding plot. The aggressive clinical course and RNA-sequencing results of the present case suggest that KMT2A-rearranged DLBCL shares biological features with KMT2A-rearranged leukemia.

Tandem high-dose chemotherapy with autologous stem cell rescue for stage M high-risk neuroblastoma: Experience using melphalan/etoposide/carboplatin and busulfan/melphalan regimens.

The efficacy of tandem HDCT against high-risk neuroblastoma has been reported; however, an optimal regimen remains to be established. In this paper, we report our experience using tandem HDCT comprising the MEC and BuMel regimens in patients with high-risk neuroblastoma. We retrospectively analyzed four patients with stage M high-risk neuroblastoma who received HDCT with MEC followed by BuMel combined with autologous stem cell rescue. Although none of their metastatic lesions had disappeared after induction chemotherapy, three patients showed a CR after tandem HDCT. Gastrointestinal mucosal injuries and renal dysfunction were observed as non-hematologic adverse events of grade 3 or higher. Gastrointestinal mucosal injuries were observed in all four patients following the first HDCT and in one patient following the second HDCT and were treated with parenteral nutrition and analgesics. One patient experienced renal dysfunction during the first HDCT, which was alleviated by sufficient hydration and diuretics and resulted in the reduction of melphalan dosage for the second HDCT. SOS was not observed in any patient. The HDCT regimens examined in this study were observed to be feasible and did not result in any life-threatening adverse events. Our findings indicate that tandem HDCT comprising MEC and BuMel is a potentially effective regimen for patients with high-risk neuroblastoma, including for those who respond poorly to induction chemotherapy, although additional studies in a larger population should be conducted to verify any long-term outcomes and toxicity.

A Japanese patient with RAD51-associated Fanconi anemia.

RAD51 is the only identified autosomal dominant gene to date causative of Fanconi anemia (FA) due to dominant negative effects. Only two patients with RAD51-associated FA have been reported with atypical FA phenotypes without bone marrow failure. We describe a new Asian patient with a novel RAD51 mutation, presenting with multiple congenital anomalies and atypical FA with chromosomal instability. The patient was a 9-year-old Japanese girl. She had strabismus, myopia, submucous cleft palate, bilateral hearing impairment, and scoliosis. She also had growth retardation, developmental delay, and severe intellectual disability. We performed trio whole exome sequencing and Sanger sequencing and identified a de novo RAD51 mutation (c.725A>G, p.Gln242Arg). Isolated lymphocytes from the patient were hypersensitive to chromosomal breakage induced by the DNA cross-linking agent, mitomycin C. Our detailed phenotypic analysis of the RAD51-associated atypical FA revealed clinical manifestations from the diverse population and a consistent FA phenotype characterized by chromosome instability, intellectual disability, radial ray abnormality, and microcephaly, but not bone marrow failure.

A case of malignant rhabdoid tumor mimicking yolk sac tumor.

Malignant rhabdoid tumors (MRTs) are rare, highly aggressive embryonal neoplasms caused by biallelic alterations of the SMARCB1 gene. MRTs may occur in any soft tissue, but extracranial extrarenal MRTs are extremely rare. Diagnosis of MRTs in unusual locations and with an uncharacteristic cytomorphology that mimics other tumors is difficult. This was an atypical case of MRT in a 15-year-old female with tumors that closely resembled yolk sac tumors. It was extremely challenging to diagnose the tumors without confirming the SMARCB1 status.

Targeted inactivation of nuclear interaction partner of ALK disrupts meiotic prophase.

NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa-/- mouse embryonic fibroblasts show no severe phenotype, Nipa-/- spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa-/- spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.

Suplatast tosilate for treating cutaneous mastocytosis.

Pediatric cutaneous mastocytosis is a rare disease caused by mast cell hyperplasia. We report the case of an infant diagnosed as cutaneous mastocytosis and seasonal allergies. The wheals, flushing, and pruritus of the mastocytosis were unresponsive to combination therapy with an antihistamine, a mast cell stabilizer (sodium cromoglycate), and a leukotriene antagonist. Addition of suplatast tosilate as a treatment for the seasonal allergy also dramatically improved his cutaneous symptoms and signs. Further trials of suplatast tosilate in selected cases of cutaneous mastocytosis are warranted.

Endoscopic Endonasal Transsphenoidal Surgery for Intrasellar Mixed Germ Cell Tumors.

A mixed germ cell tumor (MGCT) in the neurohypophysis is very rare, with only a few reported cases1-4 but none with surgical videos. In this report, the endoscopic endonasal transsphenoidal approach for MGCT in the neurohypophysis is presented (Video 1). A 12-year-old girl with ocular pain, fatigue, and nausea presented with gradual onset of quadrant hemianopsia and left oculomotor palsy. Magnetic resonance imaging showed an enhanced mass in the sella turcica with multiple components involving the pituitary gland and stalk. Her endocrinological examination showed decreased levels of pituitary hormones and simultaneously elevated serum levels of alpha-fetoprotein and beta-human chorionic gonadotropin. After pituitary hormone replacement, endoscopic endonasal transsphenoidal surgery was planned. The tumor was strongly adherent to the surrounding structures, and gross total resection was achieved. The histological diagnosis was MGCT with a teratoma component. Postoperatively, her vision and oculomotor palsy improved swiftly, and adjuvant chemotherapy and radiotherapy were administered. In this case, 3-dimensional computer graphics were created from the preoperative computed tomography and magnetic resonance imaging studies. Preoperative simulation with the 3-dimensional computer graphic images and intraoperative verification with indocyanine green images facilitated our understanding of the surrounding anatomy, including the tumor components, pituitary gland, and internal carotid arteries.5 After removal of the tumor, multilayer fascial closure was performed for skull base reconstruction.6 MGCT in the neurohypophysis can be strongly adherent to the surrounding structures, requiring careful dissection and resection under endoscopy. At the last follow-up (8 months after surgery), the tumor was successfully controlled, and the patient had no neurological symptoms with pituitary hormone replacement therapy.

RNA-seq-based miRNA signature as an independent predictor of relapse in pediatric B-cell acute lymphoblastic leukemia.

ABSTRACT: Aberrant micro-RNA (miRNA) expression profiles have been associated with disease progression and clinical outcome in pediatric cancers. However, few studies have analyzed genome-wide dysregulation of miRNAs and messenger RNAs (mRNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To identify novel prognostic factors, we comprehensively investigated miRNA and mRNA sequencing (miRNA-seq and mRNA-seq) data in pediatric BCP-ALL samples with poor outcome. We analyzed 180 patients, including 43 matched pairs at diagnosis and relapse. Consensus clustering of miRNA expression data revealed a distinct profile characterized by mainly downregulation of miRNAs (referred to as an miR-low cluster [MLC]). The MLC profile was not associated with any known genetic subgroups. Intriguingly, patients classified as MLC had significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 ×10-5). Furthermore, this poor prognosis was retained even in hyperdiploid ALL. This poor prognostic MLC profiling was confirmed in the validation cohort. Notably, non-MLC profiling at diagnosis (n = 9 of 23; Fisher exact test, P = .039) often changed into MLC profiling at relapse for the same patient. Integrated analysis of miRNA-seq and mRNA-seq data revealed that the transcriptional profile of MLC was characterized by enrichment of MYC target and oxidative phosphorylation genes, reduced intron retention, and low expression of DICER1. Thus, our miRNA-mRNA integration approach yielded a truly unbiased molecular stratification of pediatric BCP-ALL cases based on a novel prognostic miRNA signature, which may lead to better clinical outcomes.

Inhibition of the galactosyltransferase C1GALT1 reduces osteosarcoma cell proliferation by interfering with ERK signaling and cell cycle progression.

Novel therapeutic strategies are urgently required for osteosarcoma, given the early age at onset and persistently high mortality rate. Modern transcriptomics techniques can identify differentially expressed genes (DEGs) that may serve as biomarkers and therapeutic targets, so we screened for DEGs in osteosarcoma. We found that osteosarcoma cases could be divided into fair and poor survival groups based on gene expression profiles. Among the genes upregulated in the poor survival group, siRNA-mediated knockdown of the glycosylation-related gene C1GALT1 suppressed osteosarcoma cell proliferation in culture. Gene expression, phosphorylation, and glycome array analyses also demonstrated that C1GALT1 is required to maintain ERK signaling and cell cycle progression. Moreover, the C1GALT1 inhibitor itraconazole suppressed osteosarcoma cell proliferation in culture, while doxycycline-induced shRNA-mediated knockdown reduced xenograft osteosarcoma growth in mice. Elevated C1GALT1 expression is a potential early predictor of poor prognosis, while pharmacological inhibition may be a feasible treatment strategy for osteosarcoma.

Minimal residual disease detection by mutation-specific droplet digital PCR for leukemia/lymphoma.

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.

Outcomes following induction failure in Japanese children with acute lymphoblastic leukemia.

The characteristics and prognosis of Japanese children with acute lymphoblastic leukemia (ALL) who fail to achieve complete remission after remission induction chemotherapy (i.e., experience induction failure) are poorly understood. Therefore, we retrospectively analyzed data of patients enrolled in Japanese clinical trials for newly diagnosed ALL between 1996 and 2009. Among 4956 participants, 89 (1.8%) experienced induction failure. With a 6.0-year median follow-up, the 5-year overall survival rate of the entire cohort was 43.0% ± 5.5%. Survival rates did not differ between patients with B-cell precursor ALL (BCP-ALL) and T-cell ALL (T-ALL). In multivariate analysis, day 15 M3 marrow (bone marrow blast count ≥ 25%) was significantly correlated with poorer survival in the whole or BCP-ALL cohorts. In T-ALL, age < 6 years was significantly associated with poor survival. However, due to the small sample size, this correlation must be further investigated. Most T-ALL and BCR-ABL-positive BCP-ALL patients underwent allogeneic stem cell transplantation (allo-SCT). Survival rates did not differ between BCR-ABL-negative BCP-ALL patients who did and did not undergo allo-SCT, possibly due to the inclusion of lower-risk patients in the latter group. In conclusion, the induction failure rate and survival after diagnosis of induction failure in our study were comparable to previously reported figures.