Fractionated plasma N-glycan profiling of novel cohort of ATP6AP1-CDG subjects identifies phenotypic association.

ATP6AP1-CDG is an X-linked disorder typically characterized by hepatopathy, immunodeficiency, and an abnormal type II transferrin glycosylation pattern. Here, we present 11 new patients and clinical updates with biochemical characterization on one previously reported patient. We also document intrafamilial phenotypic variability and atypical presentations, expanding the symptomatology of ATP6AP1-CDG to include dystonia, hepatocellular carcinoma, and lysosomal abnormalities on hepatic histology. Three of our subjects received successful liver transplantation. We performed N-glycan profiling of total and fractionated plasma proteins for six patients and show associations with varying phenotypes, demonstrating potential diagnostic and prognostic value of fractionated N-glycan profiles. The aberrant N-linked glycosylation in purified transferrin and remaining plasma glycoprotein fractions normalized in one patient post hepatic transplant, while the increases of Man4GlcNAc2 and Man5GlcNAc2 in purified immunoglobulins persisted. Interestingly, in the single patient with isolated immune deficiency phenotype, elevated high-mannose glycans were detected on purified immunoglobulins without glycosylation abnormalities on transferrin or the remaining plasma glycoprotein fractions. Given the diverse and often tissue specific clinical presentations and the need of clinical management post hepatic transplant in ATP6AP1-CDG patients, these results demonstrate that fractionated plasma N-glycan profiling could be a valuable tool in diagnosis and disease monitoring.

Hematopoietic stem cell transplant for erythropoietic porphyrias in pediatric patients.

Cutaneous, hematopoietic, and hepatic manifestations of congenital erythropoietic porphyria (CEP) and erythropoietic protoporphyria (EPP) can be debilitating. We present our institution's experience with five patients with porphyria who underwent hematopoietic stem cell transplant (HSCT). Four patients with CEP, including three under age 2, received myeloablation. One patient with EPP, with prior liver transplant, received reduced intensity conditioning (RIC). Four patients are alive without porphyria symptomology and with full donor chimerism. HSCT corrects the defective heme pathway and should be considered early in patients with severe erythropoietic porphyrias to minimize end-organ damage. RIC regimens can minimize toxicity in patients with comorbidities.

Irradiation-free RIC HSCT has a tolerable safety profile and is effective therapy for pediatric bone marrow failure syndromes.

For patients with bone marrow failure syndromes (BMFS) who may tolerate gradual donor engraftment and achieve adequate disease control with stable mixed chimerism, RIC regimens may be preferable to myeloablative regimens. We performed a retrospective analysis of outcomes for patients who underwent HSCT at our institution between 2009 and 2017 for BMFS using an irradiation-free RIC regimen. Fourteen pediatric patients with BMFS received fludarabine (30 mg/m2 IV daily × 3), thiotepa (5 mg/kg IV every 12 hours × 2), and melphalan (70 mg/m2 IV daily × 2) prior to HSCT. Our cohort included the following diagnoses: SAA (n = 7), CAMT (n = 4), SCN (n = 1), DBA (n = 1), and non-Fanconi congenital BMF (n = 1). Seven patients underwent a MSD transplant; seven underwent an unrelated donor transplant. All patients are alive with median follow-up of 1112 days (range 455-2549 days). The median time to neutrophil engraftment was 16 days (range 10-26 days). All were transfusion independent by day + 100. The highest grade of aGVHD was grade 2; 8 (57%) did not develop aGVHD. Four (28.5%) developed extensive cGVHD, 4 (28.5%) developed limited cGVHD, and 6 (43%) did not develop cGVHD. No patients developed SOS. None died from GVHD or infectious complications. HSCT with RIC with fludarabine, thiotepa, and melphalan for BMFS was effective with a tolerable safety profile. Probability of OS at 100 days and 1 year was 100%.

Incidence of thyroid dysfunction in children after HSCT with reduced intensity conditioning (RIC) or myeloablative conditioning (MAC).

We have previously demonstrated a 11% incidence of post-transplant de novo thyroid disease, even with a radiation-free RIC regimen. Following the enactment of a universal late effects screening program at our institution, we compared the outcomes of 108 pediatric hematopoietic stem cell transplant recipients after a RIC regimen (n = 33) to those after a MAC regimen (n = 75) during the same time period. Overall, 10% of subjects developed thyroid dysfunction after HSCT, with a median follow-up of 669 days. Seven subjects had primary hypothyroidism prior to HSCT. Of the thirty-one subjects who received RIC, one (3.2%) developed a new thyroid disorder, compared to the nine of sixty-nine (13.0%) subjects who received MAC (p = .167). No significant associations were seen with donor type, graft-vs.-host disease, or total body irradiation. Nine of the 10 subjects who developed thyroid disease after transplant were asymptomatic. Continued follow-up of this contemporary cohort will further delineate risk factors for post-transplant-associated thyroid dysfunction and better inform discussions of transplant-associated sequelae.

Hematologic Manifestations of Nutritional Deficiencies: Early Recognition is Essential to Prevent Serious Complications.

Nutritional deficiencies, including deficiencies of vitamin B12, copper, and vitamin C, may result in cytopenias and hematologic symptoms. Early recognition of these deficiencies is imperative for prompt treatment and improvement in hematologic and other manifestations. We describe 5 cases which illustrate the hematologic manifestations of nutritional deficiencies and challenges to initial diagnosis and management. Supplementation of the deficient vitamin or micronutrient in all of these cases resulted in rapid resolution of cytopenias, hemorrhage, and other associated hematologic symptoms. We also review other nutritional deficiencies that manifest with hematologic symptoms and compile recommendations on treatment and expected time to response.

Third-Party and Patient-Specific Donor-Derived Virus-Specific T Cells Demonstrate Similar Efficacy and Safety for Management of Viral Infections after Hematopoietic Stem Cell Transplantation in Children and Young Adults.

Infections with double-stranded DNA viruses are a common complication after hematopoietic stem cell transplantation (HSCT) and cause significant morbidity and mortality in the post-transplantation period. Both donor-derived (DD) and third-party (TP) virus-specific T cells (VSTs) have shown efficacy and safety in viral management following HSCT in children and young adults. Owing to a greater degree of HLA matching between the recipient and stem cell donor, DD VSTs potentially persist longer in circulation compared to TP VSTs, because they are collected from a well-matched donor. However, TP VSTs are more easily accessible, particularly for smaller transplantation centers that do not have VST manufacturing capabilities, and more economical than creating a customized product for each transplant recipient. We conducted the present study to compare clinical efficacy and safety outcomes for DD VSTs and TP VSTs in a large cohort of pediatric and young adult HSCT recipients and to determine whether DD VSTs are associated with improved outcomes owing to potentially longer persistence in the recipient's circulation. This retrospective cohort study included 145 patients who received VSTs at Cincinnati Children's Hospital Medical Center (CCHMC) between 2017 and 2021 for the treatment of adenovirus, BK virus, cytomegalovirus, and/or Epstein-Barr virus. Viruses were detected using quantitative polymerase chain reaction. Patients received VSTs on a DD (NCT02048332) or TP (NCT02532452) protocol, and VST products for both protocols were manufactured in an identical fashion. The primary study outcome was clinical response to VSTs, evaluated 4 weeks after VST administration, defined as decrease in viral load to under the inclusion thresholds, or resolution of symptoms of invasive viral infection, without the need for additional conventional antiviral medication following VST administration. Secondary outcomes included graft-versus-host-disease, transplant-associated thrombotic microangiopathy, renal function, hospital length of stay, and overall survival at 30 days and 100 days after VST administration and 1 year after HSCT. Statistical analysis was performed using the Fisher exact test or chi-square test. An unpaired t test was used to compare continuous variables. The study group comprised 77 patients in the DD cohort and 68 patients in the TP cohort. Eighteen patients in the TP cohort underwent HSCT at CCHMC, and the other 50 underwent HSCT at other institutions and presented to CCHMC solely for VST administration. There was no statistically significant difference in clinical response rates between DD and TP cohorts (65.6% versus 62.7%; odds ratio [OR], 1.162; 95% confidence interval [CI], .619 to 2.164; P = .747). There were no significant differences in secondary outcomes between the 2 cohorts. The percentage of patients requiring multiple infusions for a clinical response did not differ significantly between the DD and TP cohorts (38.2% versus 32.5%; OR, .780; 95% CI, .345 to 1.805; P = .666). We found no significant difference in clinical response rate between DD VSTs and TP VSTs and a similar safety profile. Our data suggest that TP VSTs may be sufficient to control viral infection until immune reconstitution occurs despite the potential for more rapid VST clearance compared to DD VSTs. The lack of significant differences between DD VSTs and TP VSTs is an important finding, indicating that it is not necessary for every transplant center to manufacture customized DD VSTs, and that TP VSTs are a satisfactory substitute.

Scheduled administration of virus-specific T cells for viral prophylaxis after pediatric allogeneic stem cell transplant.

Infections with double-stranded DNA viruses are a significant cause of morbidity and mortality in pediatric patients following allogeneic hematopoietic stem cell transplantation (HSCT). Virus-specific T-cell therapies (VSTs) have been shown to be an effective treatment for infections with adenovirus, BK virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). To date, prophylactic regimens to prevent or mitigate these infections using conventional antiviral medications provide suboptimal response rates. Here we report on a clinical trial (NCT03883906) performed to assess the feasibility of rapid manufacturing and early infusion of quadrivalent VSTs generated from stem cell donors ("donor-derived VSTs") into allogeneic HSCT recipients with minimal or absent viremia. Patients were eligible to receive scheduled VSTs as early as 21 days after stem cell infusion. Twenty-three patients received scheduled VSTs. Twenty of 23 patients had no viremia at the time of infusion, while 3 patients had very low-level BK viremia. Two developed clinically significant graft-versus-host disease (GVHD), although this incidence was not outside of expected incidence early after HSCT, and both were successfully treated with systemic corticosteroids (n = 2). Five patients were deemed treatment failures. Three developed subsequent significant viremia/viral disease (n = 3). Eighteen patients did not fail treatment, 7 of whom did not develop any viremia, while 11 developed low-level, self-limited viremia that resolved without further intervention. No infusion reactions occurred. In conclusion, scheduled VSTs appear to be safe and potentially effective at limiting serious complications from viral infections after allogeneic transplantation. A randomized study comparing this scheduled approach to the use of VSTs to treat active viremia is ongoing.

High-Dose Methotrexate in Pediatric Acute Lymphoblastic Leukemia: Predictors of Delayed Clearance and the Effect of Increased Hydration Rate on Methotrexate Clearance.

Objectives High-dose methotrexate (HDMTX) is an important chemotherapeutic agent in the treatment of many cancers. Identification of the predictors of poor clearance during HDMTX infusions could advance the introduction of improved supportive care to prevent toxicities and reduce hospital length of stay. The purpose of this study was to identify relationships between patient physical characteristics and HDMTX clearance in the treatment of pediatric acute lymphoblastic leukemia (ALL). At our hospital, patients who have delayed methotrexate (MTX) clearance during a cycle of HDMTX receive an increased rate of hydration with subsequent cycles. This increase in hydration rate was examined for its potential to mitigate predictors of poor clearance and to prevent nephrotoxicity. Methods This study retrospectively examined the treatment records of 87 pediatric patients diagnosed with ALL who were treated on or according to Children's Oncology Group (COG) protocols AALL0232, AALL0434, AALL1131, and AALL1231. Each patient received four cycles of HDMTX (5 g/m2 over 24 hours) at two-week intervals. Patients received either 125 ml/m2/hour (standard) or 200 ml/m2/hour (delayed clearance protocol) hydration before, with, and after each infusion. MTX levels taken at 24-, 42-, and 48-hour time points were used as an indirect measure of drug clearance. Two-tailed inference for ordinary least squares regression and both heteroskedastic and paired two-tailed t-tests were performed to identify physical characteristics associated with delayed MTX clearance and the effects of hydration rate on MTX clearance, respectively. Results Patient age and body surface area (BSA) were found to have statistically significant (p<0.05) positive associations with the serum MTX levels at 24, 42, and 48 hours in cycle 1. Age and BSA were significant only at the 24-hour time point in cycles 2 and 4. Weight alone was not associated with delayed MTX clearance. For patients who had delayed MTX clearance once and thus received the delayed clearance protocol in subsequent cycles, increasing the hydration rate from 125 to 200 ml/m2/hour was associated with a statistically significant decrease in average MTX levels as well as serum creatinine levels at the 24-, 42-, and 48-hour time points. Once patients with delayed clearance received the 200 ml/m2/hour rate of hydration, the history of prior poor clearance lost its predictive value for serum MTX levels and delayed clearance. Conclusions These results suggest that patient age and BSA are significant predictors of MTX clearance if all patients receive the same rate of hydration. Age and BSA affect the distribution phase of MTX kinetics, with downstream effects in the elimination phase. Increased hydration mitigates the effects of these physical characteristics on the elimination phase kinetics by improving renal elimination of MTX, causing a loss of significance of age and BSA as predictors of MTX levels in subsequent cycles at the 42- and 48-hour time points, but with less effect at 24 hours. Thus, hyperhydration regimens prior to cycle 1 of HDMTX could be considered for patients presenting with risk factors of advanced age or high BSA to avoid delayed clearance.

Protein kinase D mediates mitogenic signaling by Gq-coupled receptors through protein kinase C-independent regulation of activation loop Ser744 and Ser748 phosphorylation.

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3-10 nm) or with vasopressin, a different G(q)-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser(744) and Ser(748). Transphosphorylation targeted Ser(744), whereas autophosphorylation was the predominant mechanism for Ser(748) in cells stimulated with G(q)-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G(q)-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G(q)-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.