We describe a 14-year-old boy who exhibited left palatine tonsillar enlargement after 6 cycles of aggressive chemotherapy for diffuse large B-cell lymphoma of the right palatine tonsil. The cervical computed tomography scan at 4 months after completion of chemotherapy revealed enlargement of the left palatine tonsil in addition to the thymus without any clinical symptoms. The F-fluorodeoxyglucose positron emission tomography indicated focal areas of strong F-fluorodeoxyglucose uptake in the left palatine tonsil. Histologic examination confirmed tonsillar hyperplasia with no evidence of recurrence. Reactive tonsillar hyperplasia after chemotherapy is rarely reported.

This study suggests the importance of the NUB domain in host defense against mycobacteria. The unique presenting features in our patient indicate that a hypomorphic NEMO mutation can be associated with atypical pathological findings of the epithelial tissues in patients with BCG infection.

Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to >50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts <50 x 103 cells/uL. Cytoskeleton function was highly abnormal in myeloid cells pre-GT, as shown by the near absence of podosome formation in monocyte-derived dendritic cells. At 12 months post-GT, the % of podosome-forming cells was improved in all subjects, and reached the level of healthy controls in the 2 patients with highest VCN in myeloid cells. Both subjects with pre-existing autoimmunity had post-GT autoimmunity: patient 4 had a flare of autoimmune cytopenias at 18 months post-GT, and patient 5 developed refractory autoimmune hepatitis and hemolytic anemia at 8 months post-GT. While all subjects had WASP expression in lymphocytes, those with autoimmunity had poor recovery of T cells, Tregs, and transitional B cells at the time of clinical symptoms. IL-10 producing regulatory B cells were deficient pre-GT and recovered to varying degrees in all subjects. No severe GT-related adverse events have occurred to date. Replication-competent lentivirus was not detected. Analysis of integration site distributions in five subjects showed reconstitution to be highly polyclonal, with no clones expanded to >20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

The role of allogeneic hematopoietic stem cell transplantation (HSCT) for infants with acute lymphoblastic leukemia (ALL) and KMT2A gene rearrangement (KMT2A-r) is controversial in terms of both its efficacy and potential of acute and late toxicities. In the Japanese Pediatric Leukemia/Lymphoma Study Group trial MLL-10, by introducing intensive chemotherapy, indication of HSCT was restricted to the patients with high-risk (HR) features only (KMT2A-r and either age &lt;180 days or presence of central nervous system leukemia). Of the 56 HR patients, 49 achieved complete remission. Forty-three patients received HSCT in first remission including the 38 patients receiving protocol-specified HSCT with conditioning consisting of individualized targeted doses of busulfan, etoposide, and cyclophosphamide. Three-year event-free survival (EFS) of 56.8% (95%CI, 42.4%-68.8%) and overall survival of 80.2% (95%CI, 67.1%-88.5%) were accomplished. Univariable analysis showed Interfant-HR criteria and flowcytometric minimal residual disease (MRD) ≥0.01% both at end of induction and at end of consolidation (EOC) were significantly associated with poorer EFS. In the multivariable analysis, positive MRD at EOC was solely associated with poor EFS (P&lt;0.001). Rapid pre-transplant MRD clearance and tailored HSCT strategy in the MLL-10 trial resulted in a favorable outcome for infants with HR KMT2A-r ALL. However, considering the high rate of potentially life-threatening toxicities and risk of late effects, its indication should be further restricted or even eliminated in the future by introducing more effective therapeutic modalities with minimal toxicities. This trial was registered at University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) as #UMIN000004801.

Cryotherapy is a conventional method for preventing melphalan‐induced oral mucositis (OM) in adult patients. We retrospectively examined the clinical benefits of cryotherapy in 41 pediatric patients undergoing autologous stem cell transplantation using a melphalan‐etoposide‐carboplatin regimen. Twenty‐two patients received cryotherapy. The cumulative incidence of grade 3‐4 OM was significantly lower in the cryotherapy group (57.1%) than in the noncryotherapy group (89.5%; P = .041). Multivariate analyses identified cryotherapy and the melphalan dose as independent factors for the lower occurrence of OM. The present study demonstrates the clinically significant efficacy of cryotherapy for preventing melphalan‐induced OM in pediatric patients.

Histiocytic sarcoma, a rare hematopoietic neoplasm with evidence of histiocytic differentiation, is often refractory to conventional chemotherapy and radiotherapy, and its prognosis is generally dismal. The optimal management of this malignancy has not been established. We report a case of 8-year-old girl with histiocytic sarcoma involving the left femur. The tumor rapidly responded to a combination of cladribine and high-dose cytosine arabinoside, an aggressive salvage regimen for refractory Langerhans cell histiocytosis, and became impalpable during the first cycle. The patient has remained in complete remission more than 7 years from diagnosis.

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