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Jonathan Greenberg,1 Jacqueline B Palmer,2 Wing W Chan,2 Catherine E Correia,1 Diane Whalley,3 Paul Shannon,3 Gregory S Sawicki1 1Division of Respiratory Diseases, Boston Children’s Hospital, Boston, MA, 2Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA; 3RTI Health Solutions, Manchester, UK Background: This study assessed treatment satisfaction of cystic fibrosis (CF) patients in a routine clinical setting for tobramycin inhalation powder (TIP), the first dry powder–inhaled antibiotic for Pseudomonas aeruginosa infection.Methods: CF patients aged 6 years or older treated with at least one cycle of TIP completed a web survey on experience with TIP, including the Treatment Satisfaction Questionnaire for Medication (TSQM). Regression analysis determined the factors associated with TSQM global satisfaction. Results: Eighty patients (mean age ± standard deviation: 24.4±9.4 years; 57.5% female; mean forced expiratory volume in 1 second ± standard deviation: 67.1%±27.3% predicted) completed the survey. The majority expressed satisfaction with TIP’s administration time (100%), time to clean (97.1%), portability (97.1%), and ease of use (94.3%). Effectiveness was significantly associated with TSQM global satisfaction (regression R-squared: 0.54). Conclusion: Patient preferences for TIP were based on administration time and ease of use. Global satisfaction was related to greater patient-perceived effectiveness. Keywords: cystic fibrosis, patient satisfaction, tobramycin, dry powder inhalers

Background: Type I glycogen storage disease (GSD) is caused by a deficiency of glucose-6-phosphatase resulting in severe fasting hypoglycemia.Objective: We compared the efficacy of a new modified starch with the currently used cornstarch therapy in patients with type Ia and Ib GSD.Design: This was a randomized, 2-d, double-blinded, crossover pilot study comparing the commonly used uncooked cornstarch with the experimental starch in 12 subjects (6 GSDIa, 6 GSDIb) aged >/= 13 y. At 2200, the subjects were given 100 g of digestible starch, and glucose and lactate were measured hourly until the subject's plasma glucose concentration reached 60 mg/dL or until the subject had fasted for 10 h. The order in which the products were tested was randomized in a blinded fashion.Results: The matched-pair Gehan rank test for censored survival was used to compare the therapies. The experimental starch maintained blood glucose concentrations significantly longer than did the traditional therapy (P = 0.013) in the 2-sided analysis. Most of the benefit was found to be after glucose concentrations fell below 70 mg/dL. The currently used cornstarch resulted in higher peak glucose concentrations and a more rapid rate of fall than did the new starch.Conclusions: The experimental starch was superior to standard therapy in preventing hypoglycemia (

Abstract Background NUP98 gene rearrangements have been reported in acute myeloid leukemia, giving rise to fusion proteins that seem to function as aberrant transcription factors, and are thought to be associated with poor prognosis. Findings A patient with treatment-related acute myeloid leukemia presented a t(3;11)(p11;p15) as the only cytogenetic abnormality. FISH and molecular genetic analyses identified a class 1 homeobox gene, POU1F1, located on chromosome 3p11, as the fusion partner of NUP98. In addition, we have found that the patient harbored an FLT3-ITD mutation, which most likely collaborated with the NUP98-POU1F1 fusion gene in malignant transformation. Conclusions We have identified POU1F1 as the NUP98 fusion partner in therapy-related AML with a t(3;11)(p11;p15). This is the first POU family member identified as a fusion partner in human cancer.

Abstract Background Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. Methods Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. Results Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. Conclusion We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene found fused with MLL in acute leukemia, future studies addressing its biologic relevance for leukemogenesis are warranted.

Abstract Background A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature. Methods Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient. Results When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023) Conclusion We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.

Background: The 2016 WHO classification described a subtype of midline gliomas harboring histone 3 (H3) K27M alterations, and the 2021 edition added a new subtype of hemispheric diffuse gliomas with H3 G34R/V mutations. The incidence and clinical behavior of leptomeningeal disease (LMD) in these patients is not well defined., Methods: Retrospective study of patients with H3-altered gliomas diagnosed from 01/2012 to 08/2021; histone mutations were identified through next-generation sequencing (NGS) of tumor biopsy and/or cerebrospinal fluid (CSF)., Results: We identified 42 patients harboring H3 mutations (K27M mutations in 33 patients, G34R/V in 8, and both in one). Median age was 21 (4-70); 27 were male. LMD was diagnosed in 21/42 (50%) patients, corresponding to a 3-year cumulative incidence of 44.7% (95% confidence interval (CI): 26.1%-63.4%) for the K27-mutant group and a 1-year cumulative incidence of 37.5% in the G34-mutant group (95% CI: 0.01%-74.4%; no events after 1 year). Median time from tumor diagnosis to LMD was 12.9 months for H3-K27 patients and 5.6 months for H3-G34 patients. H3 mutation was detected in CSF in all patients with LMD who had NGS (8 H3-K27-mutant patients). In the H3-K27-mutant group, modeled risk of death was increased in patients who developed LMD (hazard ratio: 7.37, 95% CI: 2.98-18.23, P < .0001)., Conclusions: In our cohort, 50% of patients developed LMD. Although further studies are needed, CSF ctDNA characterization may aid in identifying molecular tumor profiles in glioma patients with LMD, and neuroaxis imaging and CSF NGS should be considered for early LMD detection., Competing Interests: A.L.L has received Research funding from Bristol Myer Squib. For the remaining authors, none were declared., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Many low-grade gliomas (LGG) harbor isocitrate dehydrogenase (IDH) mutations. Although IDH mutation is known to be epileptogenic, the rate of refractory seizures in LGG with IDH mutation vs wild-type had not been previously compared. We therefore compared seizure pharmacoresistance in IDH -mutated and wild-type LGGs., Methods: Single-institution retrospective study of patients with histologic proven LGG, known IDH mutation status, seizures, and ≥2 neurology clinic encounters. Seizure history was followed until histological high-grade transformation or death. Seizures requiring ≥2 changes in anti-epileptic drugs were considered pharmacoresistant. Incidence rates of pharmacoresistant seizures were estimated using competing risks methodology., Results: Of 135 patients, 25 patients (19%) had LGGs classified as IDH wild-type. Of those with IDH mutation, 104 (94.5%) were IDH1 R132H; only 6 were IDH2 R172K. 120 patients (89%) had tumor resection, and 14 (10%) had biopsy. Initial post-surgical management included observation (64%), concurrent chemoradiation (23%), chemotherapy alone (9%), and radiotherapy alone (4%). Seizures became pharmacoresistant in 24 IDH -mutated patients (22%) and in 3 IDH wild-type patients (12%). The 4-year cumulative incidence of intractable seizures was 17.6% (95% CI: 10.6%-25.9%) in IDH -mutated and 11% (95% CI: 1.3%-32.6%) in IDH wild-type LGG (Gray's P -value = .26)., Conclusions: 22% of the IDH -mutated patients developed pharmacoresistant seizures, compared to 12% of the IDH wild-type tumors. The likelihood of developing pharmacoresistant seizures in patients with LGG-related epilepsy is independent to IDH mutation status, however, IDH -mutated tumors were approximately twice as likely to experience LGG-related pharmacoresistant seizures., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including "lactic acidosis", larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases.

Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-alpha. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver.

Objective: Multiple cerebral cavernous malformation (CCM) is the hallmark of familial presentation of cavernous malformation in the brain. We describe an ongoing Familial Cerebral Cavernous Malformation Project in the Rio de Janeiro state showing genetic profile and the pattern of emergent neuroimaging findings of this particular population besides a review of the updated recommendations for management of familial CCM versus patients harboring sporadic lesions., Method: Four families of our cohort of 9 families were genetically mapped showing mutational profile linked to CCM1. The neuroimaging paradigm was shifted from T2*gradient-echo (GRE) sequence to susceptibility weighting MR phase imaging (SWI)., Results: Only two index cases were subjected to surgery. There was no surgical intervention in any of the kindreds of our entire cohort of 9 families of our Neurovascular Program within seven years of follow-up. The genetic sequencing for mutational profile in four of these families has demonstrated only CCM1 gene affected. Our management of the familial CCM is according to the review of the literature recommendations., Conclusions: The Project of Familial Cerebral Cavernous Malformations of Rio de Janeiro detected mutations of the gene CCM1 in the first four families studied. Familial cavernous malformation are to be settled apart from the more common sporadic lesion. A set of recommendations was searched for in the literature in order to deal with these specific patients and kindreds.

Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.